Methods for detecting ascorbate in single cells

Abstract

Methods for detecting ascorbate within a cell are provided comprising: contacting the cell with a compound of Formula (I) or Formula (II); contacting the cell with an oxidant capable of oxidizing ascorbate into dehydroascorbic acid; irradiating the cell with light having a wavelength from about 400 nm to about 500 nm; and detecting fluorescence from the cell having a wavelength from about 500 nm to about 650 nm, whereupon detection of the fluorescence confirms the presence of ascorbate within the cell. Kits and additional methods associated with the above are also provided.

Description

[0001] Attorney Docket No. 103362-088WO1

[0002] METHODS FOR DETECTING ASCORBATE IN SINGLE CELLS

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] This application claims the benefit of priority to United States Provisional Patent Application No. 63 / 743,697, filed January 10, 2025, United States Provisional Patent Application No. 63 / 818,357, filed June 5, 2025, and United States Provisional Patent Application No. 63 / 865,985, filed August 18, 2025, the disclosures of which are incorporated herein by reference in their entireties.

[0005] BACKGROUND

[0006] Ascorbic acid, or vitamin C, is a micronutrient essential for redox homeostasis, barrier integrity, and iron absorption. In intracellular compartments and physiologic pH, ascorbic acid is ionized, forming ascorbate (AA). While all plants and some animals can synthesize AA, humans have lost the AA-synthesizing enzyme gulonolactone oxidase and therefore must obtain AA through a diet abundant in fresh fruits and vegetables1’2. As a water-soluble vitamin, there is no long-term storage of AA within the body, and different cell types maintain distinct AA concentrations through the expression and function of AA transporters. In humans, most cells uptake the extracellular AA via sodium-vitamin C transporter 1(SVCT1) and SVCT2, encoded by SLC23A1 and SLC23A2. In the immune system, leukocytes regulate AA levels mainly through SVCT2. SVCT1 / 2 import AA into the cell alongside sodium, and circulating leukocytes have been shown to maintain a concentration of AA 25- to 75-fold higher (2-6 mM) than the surrounding plasma (60-80 pM)3’4. However, how the high intracellular AA in leukocytes contributes to immunity remains unclear. Recent studies have shown that AA functions as a cofactor for epigenetic enzymes, including DNA and histone demethylasesr>>(). We and others showed that this AA activity is critical for promoting the differentiation of antibody-producing plasma cells in mice and humans?-'*. Correlatively, people susceptible to low AA levels, such as smokers, diabetics, and those with chronic illnesses, have increased rates of infection, suggesting that AA indeed plays an important immune role. However, despite these correlations, few mechanistic studies have been conducted to elucidateAttorney Docket No. 103362-088WO1 this connection10. One major challenge is that AA levels are heterogeneous among different cell populations, and there are currently no accessible methods to measure intracellular AA in individual cells4. Thus, there is a pressing need to develop a sensitive method to detect AA at the single-cell level.

[0007] Many current AA detection methods require a significant amount of sample or have a lower throughput capacity. For instance, AA is typically measured in the clinical setting using high-performance liquid chromatography (HPLC), followed by electrochemistry or UV detection methods11. Plate-based fluorescence biochemical assays are commonly used in basic research to quantify AA in the tissues or a cell population1-1.!, in the plate-based assay, AA is first oxidized by ascorbate oxidase or compounds like TEMPOL (4-hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl) into dehydroascorbic acid (DHA), the naturally occurring oxidized form of AA AS. The carbonyl groups on DHA condense with the diamine group on o-phenylenediamine (OPDA), forming a fluorescence compound: DHA-OPDA. However, cells must be lysed to extract the metabolites, and the concentration of AA measured represents the average of the whole population. Several fluorescence imaging and electrochemical methods have been developed to detect AA in single cells16-10. However, many of these methods are inaccessible to most researchers due to the requirement for specialized techniques or customized chemical synthesis.

[0008] There is a clear need for methods to detect ascorbate in cells, particularly at the single-cell level. This disclosure addresses this, as well as other needs.

[0009] SUMMARY

[0010] The present disclosure provides methods for detecting ascorbate within a cell. Previously, available ascorbate detection methods required a significant amount of sample or had a lower throughput capacity. In these methods, cells must be lysed to extract the metabolites, and the concentration of ascorbate measured represents the average of the whole population. The disclosed methods solve the problems associated with prior ascorbate detection methods by allowing for the detection of ascorbate at single-cell resolution, instead of providing an average among a cell or tissue population.

[0011] In some aspects, the method can include contacting the cell with a compound of Formula (I) or Formula (II):Attorney Docket No. 103362-088WO1

[0012]

[0013] wherein all variables are as defined herein.

[0014] In some aspects, the method can include contacting the cell with an oxidant capable of oxidizing ascorbate into dehydroascorbic acid. In some aspects, the method can include irradiating the cell with light having a wavelength from about 400 nm to about 500 nm. In some aspects, the method can include detecting fluorescence from the cell having a wavelength from about 500 nm to about 650 nm. In some aspects, detection of the fluorescence confirms the presence of ascorbate within the cell. In another aspect, a kit is provided. In some aspects, the kit can include a compound of Formula (I) or Formula (II) as described herein. In some aspects, the kit can include an oxidant capable of oxidizing ascorbate into dehydroascorbic acid.

[0015] In another aspect, a method is provided for measuring a concentration of ascorbate in a cell. In some aspects, the method can include contacting the cell with a compound of Formula (I) or Formula (II) as described herein. In some aspects, the method can include contacting the cell with an oxidant capable of oxidizing ascorbate into dehydroascorbic acid. In some aspects, the method can include irradiating the cell with light having a wavelength from about 400 nm to about 500 nm. In some aspects, the method can include measuring a median fluorescent intensity (MFI) of fluorescence from the cell, wherein the fluorescence has a wavelength from about 500 nm to about 650 nm. In some aspects, the MFI is correlated to the concentration of ascorbate in the cell.Attorney Docket No. 103362-088WO1 In another aspect, a method is provided for diagnosing or assessing a disease or disorder within a subject associated with a cell. In some aspects, the disease or disorder is associated with an alteration of a concentration of ascorbate within the cell compared to a healthy cell. In some aspects, the method can include contacting the cell with a compound of Formula (I) or Formula (II) as described herein. In some aspects, the method can include contacting the cell with an oxidant capable of oxidizing ascorbate into dehydroascorbic acid. In some aspects, the method can include irradiating the cell with light having a wavelength from about 400 nm to about 500 nm. In some aspects, the method can include measuring a median fluorescent intensity (MFI) of fluorescence from the cell, wherein the fluorescence has a wavelength from about 500 nm to about 650 nm. In some aspects, the MFI is correlated to the concentration of ascorbate in the cell. In some aspects, a change in the concentration of ascorbate in the cell relative to a healthy cell is indicative of the presence or state of the disease or disorder.

[0016] The details of one or more aspects of the disclosure are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the disclosure will be apparent from the description, the drawings, and the claims.

[0017] DESCRIPTION OF DRAWINGS

[0018] FIGs. 1A-1L depict and provide data regarding the development of Single-cell Ascorbate Level Sensing Assay (SALSA) as described in the examples. (FIG. 1A) Biochemical assay of AA detection. AA is chemically oxidized with 4-hydroxy-2, 2,6,6-tetramethylpiperidine 1-oxyl (TEMPOL) into dehydroascorbate (DHA), which then reacts with the diaminobenzene structure of o-phenylenediamine (OPDA) to generate a fluorescent product. The diamine group is labeled in a red circle. (FIG. 1B) OPDA and DAF-2 share a diaminobenzene group. The chemical structure of DAF-2 is shown, and the diamine group is highlighted. (FIGs. 1C-1D) Chemical structures of products for DAF-2 reaction with (FIG. 1C) nitric oxide (DAF-2 triazole or DAF-2T) or (FIG. 1D) DHA (DAF-2-DHA). (FIG. 1E) An overview of experimental design. HEK293T cells were cultured with or without 1 mM AA for 24h. (FIG. 1F) Intracellular AA level was analyzed using the biochemical assay as in (FIG. 1A). (FIG.

[0019] 1G) Increased green fluorescence signal only in the presence of TEMPOL and AA. Cells were cultured as in (FIG. 1E), labeled with DAF-2-DA, and treated with orAttorney Docket No. 103362-088WO1 without TEMPOL (TP) as indicated. Cells were analyzed by flow cytometry using the FITC channel at 515-545nm. (FIG. 1H) The principle of Single-cell Ascorbate Level Sensing Assay, or SALSA. Cells were labeled with the cell-permeable DAF-2-diacetate (DAF-2-DA) at 37°C for 10 minutes and then treated with TEMPOL at 25°C for 10 minutes. Cells were analyzed using flow cytometry. (FIGs. 1I-1J) Optimization of SALSA dye. Cells cultured with or without AA were labeled with the indicated concentrations of cell-permeable DAF-2 or DAF-FM diacetates. (FIG. 11) The median fluorescence intensity (MFI) and (FIG. 1J) the MFI ratio between cells cultured with and without AA were calculated for each condition. (FIGs. 1K-1L) Optimal DAF-2 labeling time. Cells were labeled with DAF-2-DA for either 10 or 20 minutes. Data were analyzed using (FIG. 1F) Student’s t-test where *** P < 0.001; or (FIGs. 1I-1L) two-one-way ANOVA followed by Tukey’s multiple comparisons test. Bars not sharing the same letter are significantly different (P < 0.05). Representatives of at least two independent experiments are shown.

[0020] FIGs. 2A-2O depict and provide data demonstrating how an unexpected spectral redshift significantly enhances SALSA sensitivity as described in the examples. HEK293T cells were cultured with or without AA for 24 hours and analyzed by SALSA. (FIGs. 2A-2B) TEMPOL- and AA-dependent signal has significant spillover from FITC (SALSAVerde) to PE (SALSAR°ja) channels. (FIG. 2A) FITC / PE signals from the Unstained (blue), Mock (gray), and AA (orange) groups were overlaid. Left, original signals without compensation. Right, signals in the two channels were compensated using the fluorescence from the Mock group. (FIG. 2B) Data from (FIG.

[0021] 2A) are depicted as histograms. Signals before (Uncompensated) and after compensation (Compensated) for the PE (SALSAR°ja) channel are shown. (FIG. 2C) The primary SALSA signal derived from TEMPOL and AA is red-shifted. Data was compensated as in (FIG. 2A) and (FIG. 2B), and the MFI ratio (Fold vs Mock; MFIAA / MFIMock) between cells cultured with and without AA was plotted for each open channel on a three-laser BD FACSCanto II. (FIGs. 2D-2E) Equipmentdependent enhancement of SALSA sensitivity. Cells were analyzed using SALSA with a five-laser BD FACSymphony A3. (FIG. 2D) Signals before and after compensation were shown for the BB515 (FITC equivalent; SALSAVerde) and BB630 (PE equivalent excited by the 488nm blue laser; SALSAR°ja). (FIG. 2E) The MFI ratios were calculated as in (FIG. 2C) for all available channels. (FIGs. 2F-2J) SALSA isAttorney Docket No. 103362-088WO1 quantitative and directly correlated with intracellular AA concentration. HEK293T cells were cultured with varying concentrations of AA for 24h. (FIG. 2F) Intracellular AA is quantified using the biochemical assay described in FIG. 1A. Cells were analyzed using SALSA, and the MFIs for BB515 (SALSAVerde) and BB630 (SALSAR°ia) are shown in (FIG. 2G) and (FIG. 2I). MFI was plotted against the intracellular AA concentration in (FIG. 2H) and (FIG. 2J). Linear regression was calculated, and the coefficient of determination (R2) is shown. (FIGs. 2K-2O) Establishment of standard curve. (FIG. 2K) HEK293T cells were permeabilized using streptolysin O (SLO), incubated with the indicated concentration of “spike-in” AA, sealed, followed by SALSA. (FIGs. 2L-2O) Representative histograms and graphs showing the linear correlation between SALSA signals and the spike-in AA levels. The dotted lines indicate the background levels at 1. Data were analyzed using two-one-way ANOVA followed by Tukey’s multiple comparisons test. Bars not sharing the same letter are significantly different (P < 0.05). Representatives of at least two (FIGs. 2A-2J) and four (FIGs. 2K-2O) independent experiments are shown.

[0022] FIGs. 3A-3I depict and provide data demonstrating that SALSA can specifically detect ascorbate with minimal NO interference as described in the examples. (FIG.

[0023] 3 A) AAhas no significant effect on macrophage activation. Top, Murine macrophage cell line Raw264.7 was stimulated with LPS (50 ng / mL) and IFN-g (10 ng / mL) overnight with or without AA (1 mM). Bottom, histograms showing the expression of CD80, an activation marker, and iNOS, an enzyme for NO production. (FIG. 3B) iNOS inhibition abolishes NO production. Cells were cultured as in (FIG. 3A) with or without 20pM 1400W, a selective and potent iNOS inhibitor. Nitrite ion, a proxy for NO production, was detected using the Griess assay. (FIGs. 3C-3E) SALSA enables specific detection of AA in the presence of NO. Unstimulated (top) and stimulated (bottom) Raw264.7 cells were analyzed using SALSA with a BD FACSymphony. Histograms for BB515 (SALSAVerde) and BB630 (SALSAR°ia) channels (FIG. 3C) and the MFI quantification (FIGs. 3D-3E) are shown. (FIGs. 3F-3H) SALSA can detect AA in the presence of NO. Stimulated Raw264.7 cells were treated with AA and 1400W as indicated and analyzed using SALSA. Histograms for BB515 (SALSAVerde) and BB630 (SALSAR°ia) channels (FIG. 3F) and the MFI quantification (FIGs. 3G-3H) are shown. (FIG. 3I) iNOS inhibition and NO quenching significantly improved SALSA by eliminating interference from NO. Raw264.7 cells were cultured with AAAttorney Docket No. 103362-088WO1 and stimulated as indicated for 24h. iNOS inhibitor 1400W was added throughout stimulation and NO quencher PTIO was added 30 minutes before SALSA. SALSAVerde(BB515; top panel) and SALSAR°ia(BB630; bottom panel) signals are shown. Data were analyzed using two-one-way ANOVA followed by Tukey’s multiple comparisons test. Bars not sharing the same letter are significantly different (P < 0.05). Representatives of at least two independent experiments are shown.

[0024] FIGs. 4A-4E depict and provide data regarding the identification of SVCT2 as the major ascorbate transporter in HEK293T cells using SALSA and CRIPSR as described in the examples. SVCT1 (SLC23A1) and SVCT2 (SLC23A2) were targeted in HEK293T cells using lentivirus expressing Cas9 and sgRNA. (FIG. 4A) Confirmation of sgRNA targeting efficiency. DNA flanking sgRNA-targeting sites were amplified by PCR. Amplicons were sequenced using Sanger, and the results were analyzed using TIDE assay (Tracking of Indels by Decomposition) to calculate the indel frequency. Estimated targeting efficiencies for SVCTi and SVCT2 were at least 71.7% and 69.2%, respectively. (FIG. 4B) Scheme for the internally controlled SALSA. Positive control (blue) and CRISPR-targeted cells (red) were cultured with AA for 24h. Positive control and cells from each condition were labeled antibodies against f2m, a universal antigen, conjugated with different fluorescence. The two groups of cells were mixed and underwent SALSA in the same reaction for increased consistency. (FIG. 4C) Representative FACS plots show the distinction between the mixed populations. (FIGs. 4D-4E) Mutation of SVCT2 significantly decreased intracellular AA. Histograms (FIG. 4D) and MFI quantifications (FIG. 4E) for the SALSAR°iasignal (PE) are shown. Data was analyzed using paired Student’s t-test. * P < 0.05; ns, not significant. Except for (FIG. 4A), representatives of four independent experiments are shown. Note that the results for the other two sgRNAs were similar (not shown).

[0025] FIGs. 5A-5K depict and provide data demonstrating that SALSA reveals heterogeneous ascorbate levels among blood cells as described in the examples. (FIG.

[0026] 5A) The experiment setup. Gulo-deficient mice, which cannot synthesize AA, were supplied with (AA Sufficient) or without AA (AA Deficient) for three weeks. (FIGs.

[0027] 5B-5D) Cells from blood, thymus, and spleen in AASufficientand AADeficientmice were harvested, counted (thymus and spleen), and analyzed using SALSA (FIGs. 5C-5D). Gray areas indicate background signals from DAF-2 without TEMPOL-mediated AAAttorney Docket No. 103362-088WO1 oxidation. (FIGs.5E-5G) Analysis of AA level in developing T cells. Thymocytes from AA-sufficient and -deficient mice were analyzed using SALSA. SALSA reaction without TEMPOL was shown as the background. (FIG. 5F) Gating strategy for thymocytes. DN, CD4 CD8 double negative; CD4SP, single positive. (FIG. 5G) Intracellular AA levels were inversely correlated with maturation stages. (FIGs. 5H-5K). Impact of AA deficiency on thymic populations. (FIGs. 5H-5I) Representative FACS plots for total thymocytes (FIG. 5H) and CD4SP cells (FIG. 5I). (FIGs. 5J-5K) The percentage of subsets was quantified and analyzed using two-tailed Student’s t-test. *** P < 0.001; ** P< 0.01; * P< 0.05; ns, not significant. Representative data were shown from 3 independent experiments.

[0028] FIGs. 6A-6B depict and provide data regarding the profiling of the SALSA fluorescence signals as described in the examples. (FIG. 6A) Raw264.7 and (FIG. 6B) HEK293T cells were cultured with 1 mM AA overnight and analyzed using SALSA. The fluorescence signal for each channel was compensated using the DAF-2 signal on the BB515 / FITC channel in cells cultured without AA. The red-shifted signals from DAF-2 reacted with DHA in the cells cultured with AA are plotted (orange). Unstained control (dashed line) and untreated cells (gray) were included for the fluorescence baseline. The wavelengths for excitation lasers are shown in the color bars above the histograms.

[0029] FIGs. 7A-7J depict and provide data demonstrating that SALSA is quantitative, and its signal is linearly correlated with intracellular AA as described in the examples. HEK293T (FIGs. 7A-7C) and Raw264.7 cells (FIGs. 7D-7F) were cultured with varying concentrations of AA overnight. Intracellular AA was determined using a biochemical assay, as shown in FIG. 1A (FIG.7A and 7D), and with SALSA using BD FACSCanto II (FIGs. 7B-7C and 7E-7F). For SALSA, MFIs for FITC and PE channels are shown. The MFI ratios between AA- and mock-treated cells (Fold vs Mock) were calculated and plotted against the intracellular AA concentrations. Linear regression was performed, and the coefficient of determination (R2) is shown. The dotted lines indicate the background levels at 1. Data were analyzed using one-way ANOVA with Dunnett’s post-hoc analysis against the group without AA. (FIGs. 7G-7H) Confirmation of the linear relationship between intracellular AA levels and SALSA MFIs. HEK293T cells were cultured with varying levels of AA, followed by SALSA and HPLC analysis. (FIG. 7I) Confirmation of the relationship between extracellularAttorney Docket No. 103362-088WO1 AA supplements and intracellular AA using LC-MS. HEK293T cells were cultured similarly as above with AA and the intracellular AA was analyzed using LC-MS, which validated the intracellular AA by the molecular weight. (FIG. 7J) Oxidation of AA by TEMPOL. HEK293T cells were cultured with 1 mM AA and treated with 20 mM TEMPOL for 10 minutes. Data were analyzed using two-tailed Student’s t-test. Intracellular AA was quantified using LC-MS. *** P < 0.001; ** P< 0.01; * P< 0.05; ns, not significant. Representatives of at least two independent experiments are shown.

[0030] FIGs. 8A-8H depict and provide data demonstrating that SALSA can detect ascorbate in the presence of nitric oxide as described in the examples. (FIG. 8A) The murine macrophage Raw264.7 cells were either unstimulated (Unstim.), stimulated with LPS+IFN-g (Stim.), or stimulated in the presence of iNOS inhibitor 1400W (Stim. + 1400W; 20pM) overnight. Cells were cultured in various AA concentrations (250, 62.5, 15.6, 3.9, and o pM) under each condition. NO was analyzed using the Griess assay, and the intracellular AA was analyzed using SALSA with BD FACSymphony A3. (FIG. 8B) Verification of stimulation-induced NO production. Data were analyzed using two-way ANOVA followed by Tukey’s multiple comparisons test. Bars not sharing the same letter are significantly different (P < 0.05). (FIGs. 8C-8H) Representative FACS plots and signal quantification for (FIGs. 8C, 8E, and 8G) SALSAVerdeand (FIGs. 8D, 8F, and 8H) SALSAR°ia. The MFIs for BB515 / SALSAVerdeand BB630 / SALSAR°iain response to the levels of AA supplement are shown in (FIG.

[0031] 8C) and (FIG. 8D), respectively. (FIGs. 8E-8F) The relationships between SALSA signals and AA supplement were fit using non-linear regression with the hyperbola model. The coefficients of determination (R2) are shown. Two biological replicates are included for each data point, and the Representatives of two independent experiments are shown. (FIGs. 8G-8H) Data were normalized using the MFI from cells cultured without AA (Fold background). Combined data from two experiments (n= 4 each). Data were analyzed using two-way ANOVA followed by Tukey’s multiple comparisons test, with the o pM AAfor each group as the control. *** P < 0.001; ** P< 0.01; * P< 0.05; ns, not significant.

[0032] FIGs. 9A-9B depict and provide data regarding ascorbate depletion in Gulo-KO mice. The AA-deficient Gulo-KO mice were deprived of AA supplements for one to three weeks. The AA levels in (FIG. 9A) plasma and (FIG. 9B) total splenocytes wereAttorney Docket No. 103362-088WO1 quantified using the plate-based biochemical assay. Data in (FIG. 9A) were analyzed using one-way ANOVA with Dunnett’s post-hoc analysis against the mice without AA depletion. Data in (FIG. 9B) were analyzed using two-tailed Student’s t-test. *** P < 0.001. Representatives of at least two experiments are shown.

[0033] DETAILED DESCRIPTION

[0034] The following description of the disclosure is provided as an enabling teaching of the disclosure in its best, currently known aspects. Many modifications and other aspects disclosed herein will come to mind to one skilled in the art to which the disclosed compositions and methods pertain, benefiting from the teachings presented in the descriptions herein and the associated drawings. Therefore, it is understood that the disclosures are not limited to the specific aspects disclosed and that modifications and other aspects are intended to be included within the scope of the appended claims. The skilled artisan will recognize many variants and adaptations of the aspects described herein. These variants and adaptations are intended to be included in the teachings of this disclosure and to be encompassed by the claims herein. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

[0035] As is apparent to those of skill in the art upon reading this disclosure, each of the individual aspects described and illustrated herein has discrete components and features that may be readily separated from or combined with the features of any of the other several aspects without departing from the scope or spirit of the present disclosure.

[0036] Any recited method can be carried out in the order of events recited or any other order that is logically possible. Unless otherwise expressly stated, it is in no way intended that any method or aspect set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method claim does not explicitly state in the claims or descriptions that the steps are to be limited to a particular order, it is in no way intended that an order be inferred in any respect. This holds for any possible non-express basis for interpretation, including logic concerning the arrangement of steps or operational flow, meaning derived from grammatical organization or punctuation, or the number or type of aspects described in the specification.Attorney Docket No. 103362-088WO1 All publications mentioned herein are incorporated by reference to disclose and describe the methods or materials in connection with which the publications are cited. The publications discussed herein are provided solely for their disclosure before the filing date of the present application. Further, the dates of publication provided herein can be different from the actual publication dates, which can require independent confirmation.

[0037] It is also to be understood that the terminology herein describes particular aspects only and is not intended to be limiting. Unless defined otherwise, all technical and scientific terms herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosed compositions and methods belong. It can be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the specification and relevant art and should not be interpreted in an idealized or overly formal sense unless expressly defined herein. Before describing the various aspects of the present disclosure, the following definitions are provided and should be used unless otherwise indicated. Additional terms may be defined elsewhere in the present disclosure.

[0038] Definitions

[0039] As used herein, “comprising” is interpreted as specifying the presence of the stated features, integers, steps, or components, but does not preclude the presence or addition of one or more features, integers, steps, components, or groups thereof. Moreover, each of the terms “by,” “comprising,” “comprises,” “comprised of,” “including,” “includes,” “included,” “involving,” “involves,” “involved,” and “such as” is used in its open, non-limiting sense and maybe used interchangeably. Further, the term “comprising” is intended to include examples and aspects encompassed by the terms “consisting essentially of ’ and “consisting of.” Similarly, “consisting essentially of’ is intended to include examples encompassed by the term “consisting of.” As used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context dictates otherwise.

[0040] Ratios, concentrations, amounts, and other numerical data can be expressed herein in a range format. Further, the endpoints of each of the ranges are significant both inAttorney Docket No. 103362-088WO1 relation to the other endpoint and independently of the other endpoint. There are many values disclosed herein, and each value is also disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. Ranges can be expressed herein as from “about” one particular value and to “about” another particular value. Similarly, when values are expressed as approximations, using the antecedent “about,” the particular value forms a further aspect. For example, if the value “about 10” is disclosed, then “10” is also disclosed.

[0041] When a range is expressed, a further aspect includes from the one particular value and to the other particular value. For example, where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure, e.g., the phrase “x to y” includes the range from ‘x’ to ‘y’ as well as the range greater than ‘x’ and less than ‘y’. The range can also be expressed as an upper limit, e.g. ‘about x, y, z, or less’ and should be interpreted to include the specific ranges of ‘about x,’ ‘about y,’ and ‘about z’ as well as the ranges of ‘less than x,’ ‘less than y.’ and ‘less than z.’ Likewise, the phrase ‘about x, y, z, or greater’ should be interpreted to include the specific ranges of ‘about x,’ ‘about y,’ and ‘about z’ as well as the ranges of ‘greater than x,’ greater than y,’ and ‘greater than z.’ In addition, the phrase “about ‘x’ to ‘y’,” where ‘x’ and ‘y’ are numerical values, includes “about ‘x’ to about y.”

[0042] Such a range format is used for convenience and brevity and thus, should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range but also to include all the individual numerical values or subranges encompassed within that range as if each numerical value and sub-range is explicitly recited. To illustrate, a numerical range of “about 0.1% to 5%” should be interpreted to include not only the explicitly recited values of about 0.1% to about 5%, but also include individual values (e.g., about 1%, about 2%, about 3%, and about 4%) and the sub-ranges (e.g., about 0.5% to about 1.1%; about 5% to about 2.4%; about 0.5% to about 3.2%, and about 0.5% to about 4.4%, and other possible subranges) within the indicated range.

[0043] As used herein, the terms “about,” “approximate,” “at or about,” and “substantially” mean that the amount or value in question can be the exact value or a value that provides equivalent results or effects as recited in the claims or taught herein. ThatAttorney Docket No. 103362-088WO1 is, amounts, sizes, formulations, parameters, and other quantities and characteristics are not and need not be exact but may be approximate, larger or smaller, as desired, reflecting tolerances, conversion factors, rounding, measurement error, and the like, and other factors known to those of skill in the art such that equivalent results or effects are obtained. In some circumstances, the value that provides equivalent results or effects cannot be reasonably determined. In such cases, as used herein, “about” and “at or about” mean the nominal value indicated ±10% variation unless otherwise indicated or inferred. In general, an amount, size, formulation, parameter, or other quantity or characteristic is “about,” “approximate,” or “at or about,” whether or not expressly stated to be such. Where “about,” “approximate,” or “at or about” is used before a quantitative value, the parameter also includes the specific quantitative value itself unless expressly stated otherwise.

[0044] As used herein, “optional” or “optionally” means that the subsequently described event or circumstance can or cannot occur. The description includes instances where said event or circumstance occurs and those where it does not.

[0045] As used interchangeably herein, “subject,” “individual,” or “patient” can refer to a vertebrate organism, such as a mammal (e.g., human). “Subject” can also refer to a cell, a population of cells, a tissue, an organ, or an organism, preferably to a human and constituents thereof.

[0046] Compounds are described using standard nomenclature. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which this disclosure belongs.

[0047] The compounds described herein include enantiomers, mixtures of enantiomers, diastereomers, tautomers, racemates, and other isomers, such as rotamers, as if each is specifically described unless otherwise indicated or otherwise excluded by context. It is to be understood that the compounds provided herein may contain chiral centers. Such chiral centers may be of either the R) or (S) configuration. The compounds provided herein may either be enantiomerically pure or be diastereomeric or enantiomeric mixtures. It is to be understood that the chiral centers of the compounds provided herein may undergo epimerization in vivo. As such, one of ordinary skill in the art will recognize that administering a compound in its R) form is equivalent, for compounds that undergo epimerization in vivo, toAttorney Docket No. 103362-088WO1 administering the compound in its (S) form. Unless stated to the contrary, a formula with chemical bonds shown only as solid lines and not as wedges or dashed lines contemplates each possible isomer, e.g., each enantiomer, diastereomer, and meso compound, and a mixture of isomers, such as a racemic or scalemic mixture.

[0048] Compounds described herein may contain one or more double bonds and, thus, potentially give rise to cis / trans (E / Z) isomers, as well as other conformational isomers. Unless stated to the contrary, all such possible isomers are contemplated, as well as mixtures of such isomers.

[0049] Compounds described herein may also present as an equilibrium of tautomers. For example, ketones with an a-hydrogen can exist in an equilibrium of the keto form and the enol form. Likewise, amides with an N-hydrogen can exist in an equilibrium of the amide form and the imidic acid form. Unless stated to the contrary, all possible tautomers of the compounds described herein are contemplated.

[0050] A dash (“-”) that is not between two letters or symbols is used to indicate a point of attachment for a substituent. For example, -(C=O)NH2is attached through the carbon of the keto (C=O) group.

[0051] “Halo” or “halogen” independently indicates any fluoro, chloro, bromo, or iodo. “Alkyl” is a straight chain or branched saturated aliphatic hydrocarbon group. In certain aspects, the alkyl is C1-C2, C1-C3, or Ci-Ce (i.e., the alkyl chain can be 1, 2, 3, 4, 5, or 6 carbons in length). The specified ranges as used herein indicate an alkyl group with the length of each member of the range described as an independent species. For example, Ci-Cealkyl, as used herein, indicates an alkyl group having 1, 2, 3, 4, 5, or 6 carbon atoms and is intended to mean that each of these is described as an independent species, and Ci-C4alkyl, as used herein, indicates an alkyl group having 1, 2, 3, or 4 carbon atoms and is intended to mean that each of these is described as an independent species. Examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, n-pentyl, isopentyl, tert-pentyl, neopentyl, n-hexyl, 2-methylpentane, 3-methylpentane, 2,2-dimethylbutane, and 2,3-dimethylbutane.

[0052] As used herein, “ascorbate” refers to a compound having a structure represented by the formula:Attorney Docket No. 103362-088WO1

[0053]

[0054] and is inclusive of salts and derivatives thereof. For example, ascorbate can include any of the common mineral salts of ascorbic acid, such as sodium ascorbate, which is a compound having a structure represented by the formula:

[0055]

[0056] “Dehydroascorbic acid” refers to a compound having the formula:

[0057]

[0058] OH

[0059] As used herein, the term “derivative” refers to a compound having a structure derived from the structure of a parent compound (e.g., a compound disclosed herein) and whose structure is sufficiently similar to those disclosed herein and based upon that similarity would be expected by one skilled in the art to exhibit the same or similar activities and utilities as the claimed compounds, or to induce, as a precursor, the same or similar activities and utilities as the claimed compound. Exemplary derivatives include but are not limited to, salts, esters, amides, salts of esters or amides, and N-oxides of a parent compound.

[0060] Certain materials, compounds, compositions, and components disclosed herein can be obtained commercially or readily synthesized using techniques generally known to those of skill in the art. For example, the starting materials and reagents used in preparing the disclosed compounds and compositions are either available from commercial suppliers, such as Sigma-Aldrich (formerly MilliporeSigma, Burlington, MA) or Thermo Fisher Scientific Inc. (Waltham, MA), or are prepared by methods known to those skilled in the art following procedures set forth in references such as Fieser and Fieser's Reagents for Organic Synthesis (John Wiley and Sons, 2007); Organic Reactions (John Wiley and Sons, 2004); March's Advanced OrganicAttorney Docket No. 103362-088WO1 Chemistry, (John Wiley and Sons, 8thEdition); and Larock's Comprehensive Organic Transformations (John Wiley and Sons, 3rdedition, 2017).

[0061] The present disclosure provides methods for detecting ascorbate within a cell. Previously, available ascorbate detection methods required a significant amount of sample or had lower throughput capacity. In these methods, cells must be lysed to extract the metabolites, and the concentration of ascorbate measured represents the average of the whole population. The disclosed methods solve the problems associated with prior ascorbate detection methods by allowing for the detection of ascorbate at single-cell resolution, instead of providing an average among a cell or tissue population.

[0062] In some aspects, the method can include contacting the cell with a compound of Formula I or Formula II:

[0063]

[0064] wherein:

[0065] Rlaand Rlbare independently selected from -OR4 and -NRr>aR5b;

[0066] R2aand R2bare independently selected from hydrogen and halo;

[0067] R3 is -NHR6;

[0068] R4 is independently selected at each occurrence from hydrogen and -C(O)R?;

[0069] Rv> and R5bare independently selected at each occurrence from hydrogen and Ci-Ce alkyl;

[0070] R6is selected from hydrogen and Ci-Ce alkyl; andAttorney Docket No. 103362-088WO1 R is selected from Ci-Ce alkyl and phenyl.

[0071] In some aspects, the compound of Formula (I) maybe administered as an isomeric form of Formula (I’):

[0072]

[0073] wherein all variables are as defined herein. As Formula (I’) is an isomer of Formula (I), references to Formula (I) herein are intended to cover all instances of compounds of both Formula (I) and Formula (I’).

[0074] In some aspects, the compound of Formula (II) maybe administered as an isomeric form of Formula (II’):

[0075]

[0076] wherein all variables are as defined herein. As Formula (II’) is an isomer of Formula (II), references to Formula (II) herein are intended to cover all instances of compounds of both Formula (II) and Formula (II’).

[0077] In some aspects of Formula (I), Formula (II), Formula (I’), or Formula (II’), Rlacan be -OR4. In some aspects of Formula (I), Formula (II), Formula (I’), or Formula (II’), Rlacan be -OH. In some aspects of Formula (I), Formula (II), Formula (I’), or Formula (II’), Rlacan be -OC(O)R?. In some aspects of Formula (I), Formula (II), Formula (I’), or Formula (II’), Rlacan be -OC(O)(Ci-C6 alkyl). In some aspects of Formula (I), Formula (II), Formula (I’), or Formula (II’), Rlacan be -OC(O)CH3.Attorney Docket No. 103362-088WO1 In some aspects of Formula (I), Formula (II), Formula (I’), or Formula (II’), Rlacan be -NRr>aR5b. In some aspects of Formula (I), Formula (II), Formula (I’), or Formula (II’), Rlacan be -NFL. In some aspects of Formula (I), Formula (II), Formula (I’), or Formula (II’), Rlacan be -NHRsb. In some aspects of Formula (I), Formula (II), Formula (I’), or Formula (II’), Rlacan be -N(independently Ci-Ce alkyl). In some aspects of Formula (I), Formula (II), Formula (I’), or Formula (II’), Rlacan be -N(CH3)2. In some aspects of Formula (I), Formula (II), Formula (I’), or Formula (II’), Rlacan be -N(CH2CH3)2.

[0078] In some aspects of Formula (I), Formula (II), Formula (I’), or Formula (II’), Rlbcan be -OR4. In some aspects of Formula (I), Formula (II), Formula (I’), or Formula (II’), Rlbcan be -OH. In some aspects of Formula (I), Formula (II), Formula (I’), or Formula (II’), Rlbcan be -OC(O)R?. In some aspects of Formula (I), Formula (II), Formula (I’), or Formula (II’), Rlbcan be -OC(O)(Ci-C6 alkyl). In some aspects of Formula (I), Formula (II), Formula (I’), or Formula (II’), Rlbcan be -OC(O)CH3. In some aspects of Formula (I), Formula (II), Formula (I’), or Formula (II’), Rlbcan be -NRr>aR5b. In some aspects of Formula (I), Formula (II), Formula (I’), or Formula (II’), Rlbcan be -NH2. In some aspects of Formula (I), Formula (II), Formula (I’), or Formula (II’), Rlbcan be -NHRsb. In some aspects of Formula (I), Formula (II), Formula (I’), or Formula (II’), Rlbcan be -N(independently Ci-Ce alkyl). In some aspects of Formula (I), Formula (II), Formula (I’), or Formula (II’), Rlbcan be -N(CH3)2. In some aspects of Formula (I), Formula (II), Formula (I’), or Formula (II’), Rlbcanbe -N(CH2CH3)2.

[0079] In some aspects of Formula (I), Formula (II), Formula (I’), or Formula (II’), Rlaand Rlbcan each be -OH. In some aspects of Formula (I), Formula (II), Formula (I’), or Formula (II’), Rlaand Rlbcan each be -OC(O)CH3.

[0080] In some aspects of Formula (I), Formula (II), Formula (I’), or Formula (II’), Rlaand Rlbcan each be -N(CH3)2. In some aspects of Formula (I), Formula (II), Formula (I’), or Formula (II’), Rlaand Rlbcan each be -N(CH2CH3)2.

[0081] In some aspects of Formula (I), Formula (II), Formula (I’), or Formula (II’), R2acan be hydrogen. In some aspects of Formula (I), Formula (II), Formula (I’), or Formula (II’), R2acan be halo. In some aspects of Formula (I), Formula (II), Formula (I’), orAttorney Docket No. 103362-088WO1 Formula (II’), R2acan be fluoro. In some aspects of Formula (I), Formula (II), Formula (I’), or Formula (II’), R2acan be chloro.

[0082] In some aspects of Formula (I), Formula (II), Formula (I’), or Formula (II’), R2bcan be hydrogen. In some aspects of Formula (I), Formula (II), Formula (I’), or Formula (II’), R2bcan be halo. In some aspects of Formula (I), Formula (II), Formula (I’), or Formula (II’), R2bcan be fluoro. In some aspects of Formula (I), Formula (II), Formula (I’), or Formula (II’), R2bcan be chloro.

[0083] In some aspects of Formula (I), Formula (II), Formula (I’), or Formula (II’), R2aand R2bcan each be hydrogen. In some aspects of Formula (I), Formula (II), Formula (I’), or Formula (II’), R2aand R2bcan each be fluoro. In some aspects of Formula (I), Formula (II), Formula (I’), or Formula (II’), R2aand R2bcan each be chloro.

[0084] In some aspects of Formula (I), Formula (II), Formula (I’), or Formula (II’), R3 is -NFL. In some aspects of Formula (I), Formula (II), Formula (I’), or Formula (II’), R3 is -NHR6. In some aspects of Formula (I), Formula (II), Formula (I’), or Formula (II’), R3 is -NH(CI-C6 alkyl). In some aspects of Formula (I), Formula (II), Formula (I’), or Formula (II’), R3 is -NH(CH3).

[0085] In some independent instances, R4 can be hydrogen. In some independent instances, R4 can be -C(O)R?.

[0086] In some independent instances, Rsacan be hydrogen. In some independent instances, R5acan be selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, secbutyl, t-butyl, n-pentyl, isopentyl, tert-pentyl, neopentyl, n-hexyl, 2-methylpentane, 3-methylpentane, 2,2-dimethylbutane, and 2,3-dimethylbutane.

[0087] In some independent instances, R5bcan be hydrogen. In some independent instances, R5bcan be selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, n-pentyl, isopentyl, tert-pentyl, neopentyl, n-hexyl, 2-methylpentane, 3-methylpentane, 2,2-dimethylbutane, and 2,3-dimethylbutane. In some independent instances, R6can be hydrogen. In some independent instances, R6can be selected from In some independent instances, Rsacan be hydrogen. In some independent instances, Rr>acan be selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, n-pentyl, isopentyl, tert-pentyl, neopentyl, n-hexyl, 2-methylpentane, 3-methylpentane, 2,2-dimethylbutane, and 2,3-dimethylbutane.Attorney Docket No. 103362-088WO1 In some independent instances, R7can be selected from In some independent instances, Rr>acan be hydrogen. In some independent instances, Rr>acan be selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, n-pentyl, isopentyl, tert-pentyl, neopentyl, n-hexyl, 2-methylpentane, 3-methylpentane, 2,2-dimethylbutane, and 2,3-dimethylbutane. In some independent instances, R7can be phenyl.

[0088] Representative examples of compounds of Formula (I), Formula (II), Formula (I’), or Formula (II’) that can be used include, but are not limited to:

[0089] HO OH

[0090] (DAF-FM)

[0091] (DAF-FM DA),

[0092] (DAF-2)

[0093]

[0094] (DAF-2 DA)Attorney Docket No. 103362-088WO1

[0095] (DAF-4 DA),

[0096]

[0097] (DAR-4),Attorney Docket No. 103362-088WO1

[0098] (DAR-4M), and

[0099]

[0100] (DAR-M).

[0101] In some aspects, the method can include contacting the cell with a compound of the following formula:

[0102]

[0103] (DAF-2).

[0104] In some aspects, contacting the cell with the compound of Formula (I) or Formula (II) can include contacting the cell with a solution of the compound of Formula (I) or Formula (II). In some aspects, the solution can have a concentration of the compound of Formula (I) or Formula (II) from about 1 micromolar to 20 micromolar, including exemplary values of about 1 micromolar, about 2 micromolar, about 3 micromolar, about 4 micromolar, about 5 micromolar, about 6 micromolar, about 7 micromolar, about 8 micromolar, about 9 micromolar, about 10 micromolar, about 11 micromolar, about 12 micromolar, about 13 micromolar, about 14 micromolar, about 15 micromolar, about 16 micromolar, about 17 micromolar, about 18 micromolar, about 19 micromolar, about 20 micromolar, or any subrange formedAttorney Docket No. 103362-088WO1 from the above exemplary values. In some aspects, the solution can have a concentration of the compound of Formula (I) or Formula (II) of about 10 micromolar.

[0105] In some aspects, the method can include contacting the cell with an oxidant. In some aspects, the oxidant can be capable of oxidizing ascorbate into dehydroascorbic acid. Oxidants capable of oxidizing ascorbate into dehydroascorbic acid are known in the art. In some aspects, the oxidant can include a nitroxide. Suitable nitroxides for oxidizing ascorbate into dehydroascorbic acid are known in the art. Representative examples include, but are not limited to, (4-hydroxy-2,2,6,6-tetramethylpiperidin-i-yl)oxyl (TEMPOL) and 3-(Carboxy)-2,2,5,5-tetramethyl-i-pyrrolidinyloxy, 3-Carboxy-2,2,5,5-tetramethyl-i-pyrrolidinyloxy, free radical (3-Carboxy-PROXYL). In some aspects, the oxidant can be TEMPOL.

[0106] In some aspects, contacting the cell with the oxidant can include contacting the cell with a solution of the oxidant. In some aspects, the solution can have a concentration of the oxidant from about 1 millimolar to about 50 millimolar, including exemplary values of about 1 millimolar, about 5 millimolar, about 10 millimolar, about 15 millimolar, about 20 millimolar, about 25 millimolar, about 30 millimolar, about 35 millimolar, about 40 millimolar, about 45 millimolar, about 50 millimolar, or any subrange formed from the above exemplary values. In some aspects, the solution can have a concentration of the oxidant of about 10 millimolar.

[0107] In some aspects, the method can further include contacting the cell with a nitric oxide synthase inhibitor or nitric oxide quencher. Representative examples of nitric oxide synthase inhibitors include, but are not limited to, N"-monomethyl-L-arginine (L-NMMA), N"-nitro-L-arginine (L-NNA, L-NOARG), N"-nitro-L-arginine methyl ester (L-NAME), N"-amino-L-arginine (L-NAA), N5-(i-iminoethyl)-L-ornithine (L-NIO), N"-methyl-L-arginine (L-NMA), asymmetric dimethyl-L-arginine (ADMA), monomethyl-L-arginine (L-MMA), 7-nitroindazole (7-NI), 3-bromo-7-nitroindazole, N"-propyl-L-arginine, S-methyl-L-thiocitrulline, aminoguanidine, ronopterin (VAS-203), ONO-1714, cindunistat, NCX-456, VAS-2381, GW-273629, NXN-462, CKD-712, KD-7040, and the like. Representative examples of nitric oxide quenchers include, but are not limited to, PTIO, carboxy-PTIO, and the like.Attorney Docket No. 103362-088WO1 In some aspects, the method can include irradiating the cell with light. In some aspects, the light can have a wavelength at or near an excitation maximum of the compound of Formula (I) or Formula (II). In some aspects, the light can have a wavelength from about 400 nm to about 500 nm, including exemplary values of about 400 nm, about 410 nm, about 420 nm, about 430 nm, about 440 nm, about 450 nm, about 460 nm, about 470 nm, about 480 nm, about 500 nm, or any subrange formed from the above exemplary values. In some aspects, the light can have a wavelength of 488 nm.

[0108] In some aspects, the cell can be irradiated with a light source. In some aspects, the light source can include a laser. In some aspects, the laser may emit light of a wavelength at or near the excitation maximum of the compound of Formula (I) or Formula (II). In some aspects, the laser can emit light of a wavelength at or near from about 400 nm to about 500 nm, including exemplary values of about 400 nm, about 410 nm, about 420 nm, about 430 nm, about 440 nm, about 450 nm, about 460 nm, about 470 nm, about 480 nm, about 500 nm, or any subrange formed from the above exemplary values.

[0109] In some aspects, the method can include detecting fluorescence from the cell. In some aspects, detection of the fluorescence can confirm the presence of ascorbate within the cell. In some aspects, the fluorescence can have a wavelength from about 500 nm to about 650 nm, including exemplary values of about 500 nm, about 510 nm, about 520 nm, about 530 nm, about 540 nm, about 550 nm, about 560 nm, about 570 nm, about 580 nm, about 590 nm, about 600 nm, about 610 nm, about 620 nm, about 630 nm, about 640 nm, about 650 nm, or any subrange formed from the above exemplary values.

[0110] In some aspects, the method is capable of measuring a concentration of ascorbate within the cell. In some aspects, a measurable parameter of the fluorescence can be correlated to the concentration of ascorbate in the cell. In some aspects, the measurable parameter can include a median fluorescent intensity (MFI) of the fluorescence.

[0111] The steps of irradiating the cell with a light source and / or detecting fluorescence from the cell maybe performed via an apparatus, device, or method suitable for suchAttorney Docket No. 103362-088WO1 purposes. Representative examples include via flow cytometry or fluorescence microscopy.

[0112] In some aspects, the steps or irradiating the cell with a light source and / or detecting fluorescence from the cell may be performed via flow cytometry. Flow cytometry is a laser-based analytical technique for rapidly measuring multiple physical and / or chemical characteristics of individual cells in suspension as they pass in single file through a defined interrogation region. Flow cytometry comprises introducing a sample containing cells into a hydrodynamically focused liquid stream, transporting the sample through a flow cell, and sequentially interrogating individual cells with one or more focused light sources, typically lasers. As each cell or particle passes through the interrogation region, incident light is scattered, and any fluorophores associated with the cell or particle are excited to emit fluorescent light, thereby generating optical signals characteristic of that cell or particle. When fluorescent labels are specifically bound to cellular targets, the intensity of the resulting fluorescence signal is indicative of the amount or presence of the corresponding target on or within each cell.

[0113] A flow cytometer generally includes three integrated subsystems: a fluidics subsystem that transports and focuses the sample within a sheath fluid so that cells pass the interrogation point substantially one at a time; an optical subsystem including excitation optics (e.g., lasers) and collection optics (e.g., lenses, filters, and beam splitters) configured to collect scattered and fluorescent light; and an electronics and processing subsystem that converts detected optical signals into electronic signals, digitizes these signals, and associates them with individual events for storage and analysis. The optical detection elements commonly include photomultiplier tubes and / or solid-state photodetectors such as avalanche photodiodes, which provide high-sensitivity detection of low-intensity fluorescence and scattered light signals. Each cell that traverses the interrogation region and is detected is treated as a discrete event, with the corresponding scatter and fluorescence signal intensities assigned to defined acquisition channels and recorded as multi-parameter data. Dedicated analysis software then processes the acquired event data, for example, by displaying one- or two-dimensional plots of selected parameters, applying gating strategies to define subpopulations, and computingAttorney Docket No. 103362-088WO1 statistics such as counts, frequencies, and intensity distributions for the identified subpopulations.

[0114] In some aspects, the steps or irradiating the cell with a light source and / or detecting fluorescence from the cell may be performed via fluorescence microscopy (such as confocal microscopy). Fluorescence microscopy is an optical imaging technique in which a sample containing one or more fluorescent species is illuminated with excitation light of a defined wavelength band so as to induce emission of fluorescence at a longer wavelength, and the emitted light is selectively detected to form an image of the sample. Under fluorescence microscopy, fluorophores present in or associated with the sample absorb photons from an incident excitation beam within a specified excitation wavelength range and undergo a transition to an electronically excited state. Following non-radiative relaxation, the fluorophores return to the ground state with emission of secondary radiation at a longer emission wavelength (Stokes shift), thereby generating fluorescent signal that is characteristic of the fluorophore and spatially correlated with its location in the sample. The sample is illuminated with excitation light that is spectrally selected to substantially correspond to an absorption band of the fluorophore, while an optical detection path is configured to transmit predominantly the longer-wavelength emission and to substantially block the more intense excitation radiation. As a result, the fluorescent structures appear with high contrast against a dark background, enabling sensitive detection and localization of labeled features such as cells, subcellular organelles, biomolecules, or other structures of interest.

[0115] A fluorescence microscope generally includes: an illumination source capable of providing excitation radiation, such as a mercury or xenon arc lamp, light-emitting diode, or laser, optically coupled into the microscope illumination path; an excitation filter arranged in the illumination path to restrict the excitation light to a predetermined wavelength band suitable for exciting the selected fluorophore or set of fluorophores; a dichroic (dichromatic) beamsplitter positioned at an oblique angle in the optical path, configured to reflect the excitation wavelength band toward the objective lens and to transmit at least a portion of the longer-wavelength fluorescence emission returning from the sample; an objective lens that focuses the excitation light into the sample plane and collects the emitted fluorescence for relay to the detection path; an emission (barrier) filter disposed in the detection path downstream of theAttorney Docket No. 103362-088WO1 beamsplitter, which passes the fluorescence emission band while attenuating or blocking residual excitation light and out-of-band radiation; and an image formation and detection system, such as a tube lens and eyepiece, a camera (for example, CCD, CMOS, or sCMOS), or another photosensitive detector that receives the filtered emission and produces a spatially resolved fluorescence image of the sample. In many aspects, these spectral elements (excitation filter, dichroic beamsplitter, emission filter) are integrated in a filter cube or module that can be interchanged to accommodate different fluorophores or combinations thereof.

[0116] Fluorescence microscopy can utilize intrinsic autofluorescence of sample components or, more commonly, exogenous fluorescent labels such as synthetic fluorochromes, fluorescent dyes, or genetically encoded fluorescent proteins that are specifically targeted to structures or analytes of interest. Upon excitation, only regions containing the fluorophore emit detectable fluorescence, so image contrast arises primarily from the presence, distribution, and intensity of the fluorescent signal rather than from absorption, scattering, or phase differences as in conventional brightfield microscopy. Multiple spectrally distinguishable fluorophores can be employed concurrently, with appropriate selection of excitation and emission wavelength bands, to enable simultaneous or sequential imaging of different targets within the same field of view. Fluorescence microscopy encompasses widefield illumination systems in which the entire field of the sample is illuminated and imaged in parallel, as well as confocal and related point-scanning or slit-scanning systems in which spatial filtering (for example, by a pinhole) reduces out-of-focus background and permits optical sectioning of thick specimens. Additional variants, including total internal reflection fluorescence microscopy and super-resolution localization techniques, modify the excitation geometry and / or detection strategy to restrict the excitation volume or to surpass the diffraction-limited resolution of conventional optical microscopes.

[0117] Any cell may be used in which it is desired to detect the presence of ascorbate. In some aspects, the cell may include a mammalian cell. In some aspects, the cell may be a human cell. Representative examples of human cells that may be suitable include, but are not limited to, embryonic stem cells, adult stem cells, erythrocytes, neutrophils, eosinophils, basophils, monocytes, lymphocytes, thrombocytes, megakaryocyte fragments, skeletal myocytes, cardiac myocytes, smooth muscle cells,Attorney Docket No. 103362-088WO1 osteoblasts, osteoclasts, osteocytes, bone-lining cells, chondrocytes, neurons, astrocytes, oligodendrocytes, microglia, ependymal cells, Schwann cells, satellite cells, keratinocytes, melanocytes, Merkel cells, Langerhans cells, respiratory epithelial cells, gastrointestinal epithelial cells, renal epithelial cells, glandular epithelial cells, vascular endothelial cells, lymphatic endothelial cells, fibroblasts, adipocytes, pericytes, mesangial cells, hepatic stellate cells, sperm, oocytes, Sertoli cells, granulosa cells, Leydig cells, theca cells, and the like. In some aspects, the human cell may be a cell associated with epithelial, connective, muscle, or nervous tissue.

[0118] In some aspects, the cell is associated with a disease or disorder. In some aspects, the cell can be associated with a cancer, such as a hematological malignancy, including acute myeloid leukemia. In some aspects, the cell can be associated with a metabolic disease, such as diabetes. In some aspects, the cell can be associated with immunodeficiency, such as common variable immunodeficiency.

[0119] In some aspects, the cell can be a cancer cell. In some aspects, the cancer cell maybe a leukemia cell. Representative examples of leukemias with which such a cell maybe associated, include, but are not limited to, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), hairy cell leukemia (HCL), T-cell prolymphocytic leukemia, adult T-cell leukemia, clonal eosinophilias, and transient myeloproliferative disease.

[0120] In some aspects, the cell can be provided or obtained from a sample from a subject. In some aspects, the subject is a human. The sample may be obtained from any suitable tissue or organ from the subject. In some aspects, the sample may be obtained from a tissue or organ. In some aspects, the tissue or organ is associated with or potentially associated with a disease or disorder. Representative examples of tissues or organs from which the sample can be obtained include the brain, the heart, the lungs, the liver, the kidneys, the stomach, the small intestine, the large intestine, the pancreas, the gallbladder, the esophagus, the rectum and anus, the skin, the spleen, the bladder, the uterus, the ovaries, the testes, the thyroid gland, the adrenal glands, the pituitary gland, the thymus, the spinal cord, the eyes, the ears, epithelial tissue (such as the epidermis and linings of the gut and blood vessels), connective tissue (including bone, cartilage, blood, adipose tissues, and tendons / ligaments),Attorney Docket No. 103362-088WO1 muscle tissue (including skeletal, cardiac, and smooth muscle), and nervous tissue (such as found in the peripheral nerves).

[0121] In another aspect, a method of measuring a concentration of ascorbate in a cell is provided. In some aspects, the method includes: contacting the cell with a compound of Formula (I) or Formula (II) as described herein; contacting the cell with an oxidant as described herein; irradiating the cell with light having a wavelength from about 400 nm to about 500 nm; and measuring a median fluorescent intensity (MFI) of fluorescence from the cell, wherein the fluorescence has a wavelength from about 500 nm to about 650 nm; wherein the MFI is correlated to the concentration of ascorbate in the cell.

[0122] In another aspect, a method of diagnosing or assessing a disease or disorder within a subject associated with a cell is provided, where the disease or disorder is associated with an alteration of a concentration of ascorbate within the cell compared to a healthy cell. In some aspects, the disease or disorder is associated with a decreased concentration of ascorbate within the cell compared to a healthy cell. In other aspects, the disease or disorder is associated with an increased concentration of ascorbate within the cell compared to a healthy cell. In some aspects, the cell is associated with or potentially associated with a disease or disorder. In some aspects, the method includes: contacting the cell with a compound of Formula (I) or Formula (II) as described herein; contacting the cell with an oxidant as described herein; irradiating the cell with light having a wavelength from about 400 nm to about 500 nm; and measuring a median fluorescent intensity (MFI) of fluorescence from the cell, wherein the fluorescence has a wavelength from about 500 nm to about 650 nm; wherein the MFI is correlated to the concentration of ascorbate in the cell, wherein a change in the concentration of ascorbate in the cell relative to a healthy cell is indicative of the presence or state of the disease or disorder.

[0123] In another aspect, a kit is provided. In some aspects, the kit can include a compound of Formula (I) or Formula (II) as described herein. In some aspects, the kit can include an oxidant capable of oxidizing ascorbate into dehydroascorbic acid as described herein. In some aspects, the kit can further include a nitric oxide synthase inhibitor or a nitric oxide quencher as described herein.Attorney Docket No. 103362-088WO1 In view of the described methods, below are described certain more particular aspects of the disclosure. These particularly recited aspects should not, however, be interpreted to have any limiting effect on any different claims containing different or more general teachings described herein, or that the “particular” aspects are somehow limited in some way other than the inherent meanings of the language and formulae literally used therein.

[0124] Aspect 1. A method for detecting ascorbate within a cell comprising:

[0125] contacting the cell with a compound of Formula (I) or Formula (II):

[0126]

[0127] wherein:

[0128] Rlaand Rlbare independently selected from -OR4 and -NRr>aR5b;

[0129] R2aand R2bare independently selected from hydrogen and halo;

[0130] R3 is -NHR6;

[0131] R4 is independently selected at each occurrence from hydrogen and -C(O)R?;

[0132] Rsaand R5bare independently selected at each occurrence from hydrogen and Ci-Ce alkyl;

[0133] R6is selected from hydrogen and Ci-Ce alkyl; and

[0134] R is selected from Ci-Ce alkyl and phenyl;

[0135] -contacting the cell with an oxidant capable of oxidizing ascorbate into dehydroascorbic acid;Attorney Docket No. 103362-088WO1 -irradiating the cell with light having a wavelength from about 400 nm to about 500 nm; and

[0136] -detecting fluorescence from the cell having a wavelength from about 500 nm to about 650 nm, whereupon detection of the fluorescence confirms the presence of ascorbate within the cell.

[0137] Aspect 2. The method of any aspect herein, such as aspect 1, wherein the compound of Formula (I) is administered as an isomeric form of Formula (I’):

[0138]

[0139] Aspect 3. The method of any aspect herein, such as aspect 1, wherein the compound of Formula (II) is administered as an isomeric form of Formula (II’):

[0140]

[0141] Aspect 4. The method of any aspect herein, such as any one of aspects 1-3, wherein Rlaand Rlbare each -OH.

[0142] Aspects. The method of any aspect herein, such as any one of aspects 1-3, wherein Rlaand Rlbare each -OC(O)CH3.

[0143] Aspect 6. The method of any aspect herein, such as any one of aspects 1-3, wherein Rlaand Rlbare each -N(CH3)2.

[0144] Aspect 7. The method of any aspect herein, such as any one of aspects 1-3, wherein Rlaand Rlbare each -N(CH2CH3)2.Attorney Docket No. 103362-088WO1 Aspect 8. The method of any aspect herein, such as any one of aspects 1-7, wherein R2aand R2bare each hydrogen.

[0145] Aspect 9. The method of any aspect herein, such as any one of aspects 1-7, wherein R2aand R2bare each fluoro.

[0146] Aspect 10. The method of any aspect herein, such as any one of aspects 1-7, wherein R2aand R2bare each chloro.

[0147] Aspect 11. The method of any aspect herein, such as any one of aspects 1-10, wherein Rs is -NH2.

[0148] Aspect 12. The method of any aspect herein, such as any one of aspects 1-10, Rs is -NH(CI-C6alkyl).

[0149] Aspect 13. The method of any aspect herein, such as any one of aspects 1-10, wherein Rs is -NH(CH3).

[0150] Aspect 14. The method of any aspect herein, such as aspect 1, wherein the compound of Formula (I) or Formula (II) is selected from:

[0151] (DAF-FM DA),

[0152]

[0153] (DAF-2),Attorney Docket No. 103362-088WO1

[0154] (DAF-2 DA),

[0155] (DAF-4),

[0156]

[0157] (DAR-2),Attorney Docket No. 103362-088WO1

[0158] (DAR-4),

[0159] (DAR-4M), and

[0160]

[0161] Aspect 15. The method of any aspect herein, such as aspect 1, wherein the compound of Formula (I) or Formula (II) is

[0162]

[0163] (DAF-2).

[0164] Aspect 16. The method of any aspect herein, such as any one of aspects 1-15, wherein the oxidant comprises a nitroxide.Attorney Docket No. 103362-088WO1 Aspect 17. The method of any aspect herein, such as any one of aspects 1-16, wherein the oxidant is selected from (4-hydroxy-2,2,6,6-tetramethylpiperidin-i-yl)oxyl (TEMPOL) or 3-Carboxy-2,2,5,5-tetramethyl-i-pyrrolidinyloxy, free radical (3-Carboxy-PROXYL).

[0165] Aspect 18. The method of any aspect herein, such as any one of aspects 1-17, wherein the oxidant is (4-hydroxy-2,2,6,6-tetramethylpiperidin-i-yl)oxyl (TEMPOL).

[0166] Aspect 19. The method of any aspect herein, such as any one of aspects 1-18, wherein contacting the cell with the compound of Formula (I) or Formula (II) comprises contacting the cell with a solution of the compound of Formula (I).

[0167] Aspect 20. The method of any aspect herein, such as aspect 19, wherein the compound of Formula (I) or Formula (II) has a concentration in the solution from about 1 micromolar to 20 micromolar.

[0168] Aspect 21. The method of any aspect herein, such as aspect 19 or aspect 20, wherein the compound of Formula (I) or Formula (II) has a concentration in the solution of about 10 micromolar.

[0169] Aspect 22. The method of any aspect herein, such as any one of aspects 1-21, wherein contacting the cell with the oxidant comprises contacting the cell with a solution of the oxidant.

[0170] Aspect 23. The method of any aspect herein, such as aspect 22, wherein the oxidant has a concentration in the solution from about 1 millimolar to about 50 millimolar.

[0171] Aspect 24. The method of any aspect herein, such as aspect 22 or aspect 23, wherein the oxidant has a concentration in the solution of about 20 millimolar. Aspect 25. The method of any aspect herein, such as any one of aspects 1-24, wherein the cell is irradiated with a light source.

[0172] Aspect 26. The method of any aspect herein, such as aspect 25, wherein the light source comprises a laser.

[0173] Aspect 27. The method of any aspect herein, such as any one of aspects 1-26, wherein the cell is irradiated with light having a wavelength of 488 nm.Attorney Docket No. 103362-088WO1 Aspect 28. The method of any aspect herein, such as any one of aspects 1-27, wherein irradiating the cell and / or detecting the fluorescence occurs via flow cytometry.

[0174] Aspect 29. The method of any aspect herein, such as any one of aspects 1-27, wherein irradiating the cell and / or detecting the fluorescence occurs via fluorescence microscopy.

[0175] Aspect 30. The method of any aspect herein, such as any one of aspects 1-29, further comprising contacting the cell with a nitric oxide synthase inhibitor or nitric oxide quencher prior to irradiating the cell with light.

[0176] Aspect 31. The method of any aspect herein, such as any one of aspects 1-30, wherein the method detects a concentration of ascorbate within the cell.

[0177] Aspect 32. The method of any aspect herein, such as aspect 31, wherein a median fluorescent intensity (MFI) of the fluorescence is correlated to the concentration of ascorbate in the cell.

[0178] Aspect 33. The method of any aspect herein, such as any one of aspects 1-32, wherein the cell is a mammalian cell.

[0179] Aspect 34. The method of any aspect herein, such as any one of aspects 1-33, wherein the cell is a human cell.

[0180] Aspect 35. The method of any aspect herein, such as any one of aspects 1-34, wherein the cell is associated with a disease or disorder.

[0181] Aspect 36. The method of any aspect herein, such as aspect 35, wherein the disease or disorder comprises a hematological malignancy, a metabolic disorder, or an immunodeficiency.

[0182] Aspect 37. The method of any aspect herein, such as aspect 35 or 36, wherein the disease or disorder is associated with a decreased concentration of ascorbate within the cell compared to a healthy cell.

[0183] Aspect 38. The method of any aspect herein, such as aspect 35 or 36, wherein the disease or disorder is associated with an increased concentration of ascorbate within the cell compared to a healthy cell.Attorney Docket No. 103362-088WO1 Aspect 39. The method of any aspect herein, such as any one of aspects 1-38, wherein the cell is a blood cell.

[0184] Aspect 40. The method of any aspect herein, such as any one of aspects 1-38, wherein the cell is an immune cell.

[0185] Aspect 41. The method of any aspect herein, such as any one of aspects 1-38, wherein the cell is a cancer cell.

[0186] Aspect 42. The method of any aspect herein, such as aspect 41, wherein the cancer cell is a leukemia cell.

[0187] Aspect 43. The method of any aspect herein, such as any one of aspects 1-42, wherein the cell is provided in a sample from a subject.

[0188] Aspect 44. The method of any aspect herein, such as aspect 43, wherein the sample is a blood sample, a bone marrow sample, a tumor sample, a tonsil sample, a spleen sample, or a lymph node sample.

[0189] Aspect 45. The method of any aspect herein, such as aspect 43 or 44, wherein the subject is a human.

[0190] Aspect 46. A kit comprising:

[0191] a compound of Formula (I) or Formula (II):

[0192]

[0193] wherein:

[0194] Rlaand Rlbare independently selected from -OR4 and -NRr>aR5b;Attorney Docket No. 103362-088WO1 R2aand R2bare independently selected from hydrogen and halo;

[0195] R3 is -NHR6;

[0196] R4 is independently selected at each occurrence from hydrogen and -C(O)R?;

[0197] R5aand R5bare independently selected at each occurrence from hydrogen and Ci-Ce alkyl;

[0198] R6is selected from hydrogen and Ci-Ce alkyl; and

[0199] R is selected from Ci-Ce alkyl and phenyl; and

[0200] an oxidant capable of oxidizing ascorbate into dehydroascorbic acid.

[0201] Aspect 47. The kit of any aspect herein, such as aspect 46, wherein the compound of Formula (I) is provided as an isomeric form of Formula (I’):

[0202]

[0203] Aspect 48. The kit of any aspect herein, such as aspect 46, wherein the compound of Formula (II) is provided as an isomeric form of Formula (II’):

[0204]

[0205] Aspect 49. The kit of any aspect herein, such as any one of aspects 46-48, wherein Rlaand Rlbare each -OH.

[0206] Aspect 50. The kit of any aspect herein, such as any one of aspects 46-48, wherein Rlaand Rlbare each -OC(O)CH3.Attorney Docket No. 103362-088WO1 Aspect 51. The kit of any aspect herein, such as any one of aspects 46-48, wherein Rlaand Rlbare each -N(CH3)2.

[0207] Aspect 52. The kit of any aspect herein, such as any one of aspects 46-48, wherein Rlaand Rlbare each -N(CH2CH3)2.

[0208] Aspect 53. The kit of any aspect herein, such as any one of aspects 46-52, wherein R2aand R2bare each hydrogen.

[0209] Aspect 54. The kit of any aspect herein, such as any one of aspects 46-52, wherein R2aand R2bare each fluoro.

[0210] Aspect 55. The kit of any aspect herein, such as any one of aspects 46-52, wherein R2aand R2bare each chloro.

[0211] Aspect 56. The kit of any aspect herein, such as any one of aspects 46-55, wherein Rs is -NFL.

[0212] Aspect 57. The kit of any aspect herein, such as any one of aspects 46-55, R3 is -NH(CI-C6alkyl).

[0213] Aspect 58. The kit of any aspect herein, such as any one of aspects 46-55, wherein R3 is -NH(CH3).

[0214] Aspect 59. The kit of any aspect herein, such as aspect 46, wherein the compound of Formula (I) or Formula (II) is selected from:

[0215] HO OH

[0216] H3CF

[0217] (DAF-FM)

[0218]

[0219] (DAF-FM DA),Attorney Docket No. 103362-088W01

[0220] (DAF-2 DA),

[0221]

[0222] Attorney Docket No. 103362-088WO1

[0223] (DAR-4),

[0224] (DAR-4M), and

[0225]

[0226] Aspect 60. The kit of any aspect herein, such as aspect 46, wherein the compound of Formula (I) or Formula (II) isAttorney Docket No. 103362-088WO1

[0227]

[0228] Aspect 61. The kit of any aspect herein, such as any one of aspects 46-60, wherein the oxidant comprises a nitroxide.

[0229] Aspect 62. The kit of any aspect herein, such as any one of aspects 46-61, wherein the oxidant is selected from (4-hydroxy-2,2,6,6-tetramethylpiperidin-i-yl)oxyl (TEMPOL) or 3-Carboxy-2,2,5,5-tetramethyl-i-pyrrolidinyloxy, free radical (3-Carboxy-PROXYL).

[0230] Aspect 63. The kit of any aspect herein, such as any one of aspects 46-62, wherein the oxidant is (4-hydroxy-2,2,6,6-tetramethylpiperidin-i-yl)oxyl (TEMPOL).

[0231] Aspect 64. The kit of any aspect herein, such as any one of aspects 46-63, further comprising a nitric oxide synthase inhibitor or nitric oxide quencher.

[0232] Aspect 65. The kit of any aspect herein, such as any one of aspects 46-64, wherein the compound of Formula (I) or Formula (II) is provided as a solution of the compound of Formula (I).

[0233] Aspect 66. The kit of any aspect herein, such as aspect 65, wherein the compound of Formula (I) or Formula (II) has a concentration in the solution from about 1 micromolar to 20 micromolar.

[0234] Aspect 67. The kit of any aspect herein, such as aspect 65 or aspect 66, wherein the compound of Formula (I) or Formula (II) has a concentration in the solution of about 10 micromolar.

[0235] Aspect 68. The kit of any aspect herein, such as any one of aspects 46-67, wherein the oxidant is provided as a solution of the oxidant.

[0236] Aspect 69. The kit of any aspect herein, such as aspect 68, wherein the oxidant has a concentration in the solution from about 1 millimolar to about 50 millimolar. Aspect 70. The kit of any aspect herein, such as aspect 68 or aspect 69, wherein the oxidant has a concentration in the solution of about 20 millimolar.Attorney Docket No. 103362-088WO1 Aspect 71. A method for measuring a concentration of ascorbate in a cell comprising:

[0237] -contacting the cell with a compound of Formula (I) or Formula (II):

[0238]

[0239] wherein:

[0240] R1aand R1bare independently selected from -OR4 and -NR5aR5b;

[0241] R2aand R2bare independently selected from hydrogen and halo;

[0242] R3 is -NHR6;

[0243] R4 is independently selected at each occurrence from hydrogen and -C(O)R7;

[0244] R5aand R5bare independently selected at each occurrence from hydrogen and C1-C6 alkyl;

[0245] R6is selected from hydrogen and C1-C6 alkyl; and

[0246] R is selected from C1-C6 alkyl and phenyl;

[0247] -contacting the cell with an oxidant capable of oxidizing ascorbate into dehydroascorbic acid;

[0248] -irradiating the cell with light having a wavelength from about 400 nm to about 500 nm; and

[0249] -measuring a median fluorescent intensity (MFI) of fluorescence from the cell, wherein the fluorescence has a wavelength from about 500 nm to about 650 nm;Attorney Docket No. 103362-088WO1 wherein the MFI is correlated to the concentration of ascorbate in the cell. Aspect 72. A method for diagnosing or assessing a disease or disorder within a subject associated with a cell, where the disease or disorder is associated with an alteration of a concentration of ascorbate within the cell compared to a healthy cell, the method comprising:

[0250] -contacting the cell with a compound of Formula (I) or Formula (II):

[0251]

[0252] wherein:

[0253] R1aand R1bare independently selected from -OR4 and -NR5aR5b;

[0254] R2aand R2bare independently selected from hydrogen and halo;

[0255] R3 is -NHR6;

[0256] R4 is independently selected at each occurrence from hydrogen and -C(O)R7;

[0257] R5aand R5bare independently selected at each occurrence from hydrogen and C1-C6 alkyl;

[0258] R6is selected from hydrogen and C1-C6 alkyl; and

[0259] R is selected from C1-C6 alkyl and phenyl;

[0260] -contacting the cell with an oxidant capable of oxidizing ascorbate into dehydroascorbic acid;

[0261] -irradiating the cell with light having a wavelength from about 400 nm to about 500 nm; andAttorney Docket No. 103362-088WO1 -measuring a median fluorescent intensity (MFI) of fluorescence from the cell, wherein the fluorescence has a wavelength from about 500 nm to about 650 nm; wherein the MFI is correlated to the concentration of ascorbate in the cell, and wherein a change in the concentration of ascorbate in the cell relative to a healthy cell is indicative of the presence or state of the disease or disorder.

[0262] A number of aspects of the disclosure have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other aspects are within the scope of the following claims.

[0263] By way of non-limiting illustration, examples of certain aspects of the present disclosure are given below.

[0264] EXAMPLES

[0265] The following examples are set forth below to illustrate the methods claimed herein, along with associated methods and results according to the disclosed subject matter. These examples are not intended to be inclusive of all aspects of the subject matter disclosed herein, but rather to illustrate representative methods and results. These examples are not intended to exclude equivalents and variations of the present disclosure, which are apparent to one skilled in the art.

[0266] Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C or is at ambient temperature, and pressure is at or near atmospheric.

[0267] SALSA: A Flow Cytometry Assay to Detect Ascorbate at Single-Cell Level Ascorbate (AA) is an essential antioxidant and enzymatic cofactor with emerging roles in epigenetic regulation, redox biology, and immune function. However, singlecell quantification of intracellular AA has remained technically challenging. In this example, we present SALSA (Single-cell Ascorbate Level Sensing Assay), a flow cytometry-based method that enables sensitive, specific detection of intracellular AA at the single-cell level. Inspired by the mechanism of the in vitro AA assay, we identified 4,5-diaminofluorescein (DAF-2), a common nitric oxide (NO) probe, as aAttorney Docket No. 103362-088WO1 selective AA reporter. We showed that the chemical oxidation of AA into dehydroascorbic acid (DHA) facilitated its reaction with DAF-2 to form a highly fluorescent product. Surprisingly, the DAF-2-DHA adduct exhibits a red-shifted emission spectrum distinguishable from those of DAF-2 alone or its NO-reactive product. This spectral shift enables the differentiation of signals into two channels, SALSAVerde(green) and SALSARoja(red-orange), with SALSARojaoffering superior sensitivity and minimal NO interference. SALSA is quantitative, with a strong linear correlation between signal intensity and intracellular AA concentration. Using SALSA and CRISPR, we identified SVCT2 as the major AA transporter in a human cell line model. Applying SALSA to immune profiling revealed previously unappreciated heterogeneity in AA levels across immune subsets and developmental stages. Together, these findings establish SALSA as a robust and accessible method for probing AA dynamics at single-cell resolution, with broad potential applications in redox biology, immunology, and metabolism.

[0268] To overcome the issues with prior AA assays, we developed the Single-cell Ascorbate Level Sensing Assay (SALSA), an accessible, sensitive, and high-throughput cytometer-based method capable of detecting AA in single cells. Based on the biochemical reaction between DHA and OPDA, we searched for fluorescence compounds with the diamine group that could react with DHA. We identified diaminofluorescein-2 (DAF-2), a common nitric oxide (NO) indicator with a similar diamine structure that reacts with NO to form the fluorescent DAF-2 -triazole (DAF-2T)20. We showed that DAF-2 fluorescence intensity increased significantly only in cells cultured with AA after TEMPOL treatment, consistent with previous studies demonstrating the reaction between DAF-2 and DHA1^21. Surprisingly, compared to DAF-2 and DAF-2T, DAF-2-DHA has a red-shifted emission spectrum that makes it selectively quantifiable by flow cytometers. SALSA is quantitative; its signal has an excellent linear correlation with intracellular AA concentrations, and it is possible to generate cells with defined AA concentrations to establish a standard curve for absolute quantification. We showed that the spectral shift enables the detection of AA despite the presence of NO, and interference from NO can be mitigated by incorporating NO quenchers and inhibitors for the nitric oxide synthases. Using SALSA, we performed a proof-of-concept CRISPR screen and identified SVCT2 as the main AA transporter in HEK293T cells. We further demonstrated theAttorney Docket No. 103362-088WO1 heterogeneous AA levels across different hematopoietic cells and differentiation stages. In summary, SALSA offers a sensitive yet specific method for detecting intracellular AA on a per-cell basis. The ability to quantify and differentiate cells by intracellular AA levels or AA transporter activity highlights SALSA’s applications in functional genomics and cellular metabolism studies.

[0269] MATERIALS AND METHODS

[0270] Cell lines. HEK293T (including WT and CRISPR-targeted) and Raw264.7 cells were cultured in DMEM and RPMI1640, respectively (Gibco). All media were supplemented with GlutaMAX (2 mM, Gibco), 10% fetal bovine serum (Gemini), HEPES (10 mM, Gibco), and Gentamicin (10 pg / mL, Gibco). Cells were cultured at 37°C with 5% CO2. Cells were regularly tested for mycoplasma contamination and were negative.

[0271] Animal Experiments. All animal experiments have been approved by IACUC committee at Ohio State University (2020A00000055-R1; approved on 8 / 8 / 2023). Gulo-deficient mice (6-12 weeks) were maintained using water containing 1 g / L AA (sodium L-ascorbate, Sigma) and 10 pM ethylenediaminetetraacetic acid (EDTA, Sigma). Water was changed weekly. To deplete AA, the mice were fed regular water without AA for three weeks. Both sexes of mice were used. Spleens, thymuses, and blood were obtained and analyzed as previously described²³.

[0272] Plate-based ascorbate assay. HEK293T cells were plated at 5x105 cells per well in a 6-well plate with or without L-ascorbic acid (AA, Sigma-Aldrich) for 16-24 hours at 37°C and 5% CO2. 2-phospho-ascorbic acid (Sigma-Aldrich) was used for Raw264.7 stimulation experiments, as AA interferes with the Griess reaction for NO detection. In vitro cultured cells or splenocytes were washed twice with phosphate-buffered saline (PBS), collected into 1.5 mL Eppendorf tubes, counted, and centrifuged at 500 xg for 5 minutes at 4 °C. Cell pellets were resuspended in a final concentration of 1.67% perchloric acid with 1 mM DTPA (diethylenetriaminepentaacetic acid, Thermo Fisher Scientific) to extract the metabolites and stabilize AA. Lysate was vortexed at high speed for 30 seconds, followed by centrifugation at 13,000 xg for 5 minutes at 4°C. Supernatants were then plated for AA assay or stored at -80°C for later use. Samples or AA standards (50 pL) were plated in a black 96-well micro titer plate (Thermo Fisher Scientific). To oxidize AA in the sample, 50 pL of 2.32 mMAttorney Docket No. 103362-088WO1 TEMPOL (4-hydroxy-2,2,6,6-tetramethylpiperidine 1-Oxyl, Tokyo Chemical Industry) in AA assay buffer (2 M sodium acetate, pH 5.5) was added to the samples, followed by 10 minutes of incubation at room temperature. To develop the fluorescence signal, 50 pL of 5 mM OPDA (o-phenylenediamine, Sigma Aldrich) in AA assay buffer was added, followed by 5 minutes of incubation at room temperature. The fluorescence signal was measured using a SpectraMax i3x plate reader (Molecular Devices) with an excitation wavelength of 345 nm and an emission wavelength of 425 nm. All procedures were performed in the dark. The intracellular concentrations were calculated using the cell volume derived from the measured average diameters of HEK293T (10-12.5 pm) and splenocytes (5 pm) using a Corning Cell Counter (Corning).

[0273] Macrophage stimulation. Raw264.7 cells were plated at a density of 5x105 cells per well in a 6-well plate (Corning) for 16-24 hours. To induce NO production, cells were stimulated with murine interferon-y (10 ng / mL, Peprotech) and lipopolysaccharides (LPS; 50 ng / mL, Sigma) for 16-24 hours at 37°C and 5% CO2.

[0274] Griess reaction. Nitrite levels in the samples were quantified using the Griess Reagent Kit (Thermo Fisher Scientific), following the manufacturer’s protocol. Briefly, 130 pL of distilled water and 150 pL of sample or standard were loaded. Griess reagent was freshly prepared by mixing N-(1-naphthyl)ethylenediamine (Component A) and sulfanilic acid (Component B) in a 1:1 ratio, and 20 pL of the Griess reagent was added to each well. Plates were incubated at room temperature for 30 minutes in the dark to allow chromophore development. Absorbance was measured at 548 nm using a SpectraMax i3x plate reader (Molecular Devices). A standard curve was generated using sodium nitrite, and nitrite concentrations in the samples were calculated based on the absorbance values. All measurements were performed in triplicate to ensure reproducibility.

[0275] Lentiviral CRISPR targeting. A lentiviral CRISPR-Casq system based on lentiCRISPR v2 (Addgene 52961) was used to mutate the vitamin C transporters, SLC23A1 and SLC23A2->. Single-guide RNAs (sgRNAs) were designed using ChopChop28. To generate the virus, the targeting vectors (500 ng) expressing sgRNA were co-transfected with the packaging vectors psPax2 (375 ng, Addgene 12260) and pMD2. G (125 ng, Addgene 12259). The virus was collected 48 hours post-transfection and used to transduce fresh HEK293T cells with polybrene (8 pg / mL, Millipore).Attorney Docket No. 103362-088WO1 Transduced HEK293T cells were selected using puromycin (1 ug / mL) and cultured for at least seven days before analysis.

[0276] Verification of CRISPR targeting. Genomic DNA (gDNA) from control and CRISPR-targeted HEK293T cells was isolated using the NucleoSpin Tissue kit (Macherey-Nagel). Purified DNA was measured with the Qubit™ ssDNA Assay Kit (Thermo Fisher Scientific, Q10212). PCR reactions were carried out with 100 ng of gDNA from each condition using the NEBNext Q5 polymerase (NEB, M0544L) with the high-fidelity protocol. PCR conditions were 30s at 98°C (lx), followed by 10s at 98°C, 10s at 64°C, and 30s at 72°C (35 cycles). Amplicon sizes (~500bp) were verified using DNA gel electrophoresis on a 1.5% agarose gel. PCR products were purified using 2x volume of solid-phase reversible immobilization (SPRI) beads followed by standard protocol (prepared in-house). Purified PCR amplicons were Sanger Sequenced with forward and reverse primers separately. Sequencing results were analyzed using Tracking of Indels by Decomposition (TIDE) to determine the spectrum and frequency of CRISPR / Cas9-induced insertions and deletions as described³⁰. Within the expected break site, reference sequences are compared to DNA traces for indels by signal decomposition, and the “% aberrant sequence” is obtained per position.

[0277] Antibodies and Flow Cytometry. HEK293T cells, including wild-type, vector control, and CRISPR / Cas9-generated knockout lines for SVCT1 and SVCT2, were treated with 1 mM AA for 24 hours at 37°C with 5% CO2. After treatment, cells were harvested and labeled with either APC- (BioLegend, 395711, 1:400) or Pacific Blue-anti-p2M antibody (BioLegend, 395731, 1:400) at 4°C for 30 minutes. After washing, cells were combined and analyzed using SALSA. For Raw264.7, cells were washed, blocked with anti-Fc receptor antibodies (BioXCell, 10 min, 4°C), and stained for macrophage markers BV650-anti-CD11b (BioLegend, 101259, 1:200), BV421-anti-CD80 (BioLegend, 104726, 1:200), PE-Cy7-anti-F4 / 80 (BioLegend, 123114, 1:200), and Zombie UV Viability Dye for live dead stain (BioLegend, 423108, 1:2000). Cells from Gulo mice were stained with BUV661 anti-CD11b (BD, 612977, 1:400), BUV737 anti-CD25 (BD, 569600, 1:400), BUV805 anti-CD45.2 (BD, 569200, 1:200), BV605 anti-CD8a (Biolegend, 100744, 1:400), BV785 anti-CD44 (Biolegend, 103059, 1:500), Pacific Blue anti-CD19 (Biolegend, 115523, 1:100), RB705 anti-CD62L (BD, 570283, 1:500), SparkPlus UV395 anti-CD4 (Biolegend, 100496, 1:400), and ZombieAttorney Docket No. 103362-088WO1 UV Viability Dye. Cells were fixed with 1% paraformaldehyde at room temperature for 20 minutes, permeabilized with lx Perm buffer containing 0.5% saponin (Intracellular Staining Permeabilization Wash Buffer, BioLegend), and incubated with PE-anti-N0S2 (BioLegend, 696806, 1:1000) antibodies for 30 minutes at 4°C in the dark. After three washes with Perm buffer, cells were resuspended in FACS buffer and analyzed using a flow cytometer. Cells were kept on ice in the dark for SALSA samples and analyzed immediately. All samples were analyzed using FACSCanto II or FACSymphony A3 (BD Bioscience).

[0278] SALSA. Cells were washed twice with PBS to remove any residual AA and resuspended in 100 pL of plain DMEM (Gibco) with 10 pM DAF-2-DA (Thermo Fisher Scientific, 50-596-008) and eFluor 780 Viability Dye (eBioscience, 65-0865-14, 1:1000), followed by 10 minutes incubation at 37°C. Ascorbate was then oxidized with 100 pL of 20 mM TEMPOL in plain DMEM at room temperature for 10 minutes. Cells were washed twice with cold FACS buffer (1% bovine serum albumin, 0.05% sodium azide, and 1 mM EDTA in PBS) and analyzed immediately. Due to the light sensitivity of DAF-2, all procedures were performed in the dark. To ensure staining quality, DAF-2-DA was thawed no more than twice, and TEMPOL was made fresh for each experiment. After labeling, the samples should be kept on ice in the dark and analyzed within 1 hour for consistency. To analyze and quantify the spectral red-shift of DAF-2-DHA relative to DAF-2 or DAF-2T, cells labeled with DAF-2 alone without AA- and TEMPOL-treatment were used as a single-color control to calculate the spectral spillover from SALSAVerde(FITC / BB515) to the SALSARoja(PE / BB630) and other channels. Cells labeled with DAF-2 and treated with AA and TEMPOL are used as a single-color control for SALSA using the SALSAR°iachannel. Compensation for SALSARojais manually adjusted to subtract its spillovers to other non-SALSA channels.

[0279] Reversible permeabilization and AA loading. Cells were washed once with calcium-free PBS and dissociated using 0.05% trypsin. Following neutralization, cells were washed with HBSS (Corning), counted, resuspended in 250 pL HBSS, and aliquoted into 1.5 mL tubes. Streptolysin O (SLO; Thermo Fisher Scientific, 50-177-9236) was diluted in HBSS to the indicated concentrations (200 ng / mL), and 250 pL of SLO or HBSS was added to the cells followed by 10 minutes incubation at 37°C. The SLO concentration was titrated to maximize the percentage of permeabilizedAttorney Docket No. 103362-088WO1 cells while minimizing cell death. To monitor permeabilization, eFluor 780 Viability Dye was added during SLO treatment. On average, about 30-60% of cells were permeabilized, as indicated by eFluor 780. Immediately following incubation, cells were placed on ice and treated with AA for 10 minutes. After centrifugation, the permeabilization buffer was removed, and samples were washed once with HBSS. To initiate membrane resealing, 1 mL of complete DMEM was added, followed by 30 minutes of incubation on ice. Cells were then washed with HBSS and stained with 7-Aminoactinomycin D (7-AAD; BioLegend, 420404) to label dead cells for 10 minutes at room temperature. Excess dye was removed by washing once with MACS buffer, and cells were resuspended in HBSS and processed for SALSA labeling as described above.

[0280] Quantification of L-ascorbic acid using HPLC-MS. One million cells were washed twice with PBS and resuspended in 100 pL of cold HPLC-grade methanol (Thermo Scientific, A456) with 0.1% formic acid (Thermo Scientific, A117), followed by vertexing for 2 minutes to lyse the cells. The homogenates were placed at -20°C for 20 minutes and then centrifuged at 14,000 rpm for 10 minutes at 4°C. The supernatant was transferred to LC vials and stored at -80°C for further LC-MS. To measure the LAA concentrations in samples, a standard curve was prepared using eight concentrations of LAA: 3.9 pM, 7.8 pM, 15.6 pM, 31.3 pM, 62.5 pM, 125 pM, 250 pM, and 500 pM. The chromatographic separation was performed by using an Atlantis T3 Colum (100A, 3 pm, 2.1 mm X 100 mm, Waters, SKU: 186003718) with the HPLC system (Thermo Scientific, UltiMate™ 3000). Metabolites were eluted from the column using a gradient mobile phase that consisted of phase A (Water with 0.1% Formic Acid, Thermo Scientific, LS118) and phase B (Acetonitrile with 0.1% Formic Acid, Thermo Scientific, LS120) at a flow rate of 0.2 mL / min. A volume of 5 pL per sample and the linear gradient elution procedure was set as follows: 0% B for 0-1 minute, from 0% B at 1 minute to 100% B at 5 minute until 7 minute, then returning to 0% at 9 minute, followed by maintaining 0% B for 9-10 minute. Column temperature was kept at 40 °C during the HPLC separation. The mass spectra were collected on a Thermo Scientific Q Exactive™ Plus mass spectrometer in negative mode. Specifically, MSi data were collected at a resolution of 70,000, with a chromatographic peak width of 6 seconds, an automatic gain control (AGC) target of ie5, a maximum injection time of 200 ms, and a scan range of 60-900 m / z. MS2 dataAttorney Docket No. 103362-088WO1 for LAA in all samples were collected at a resolution of 17,500, with an AGC target of 2e5, a maximum injection time of 100 ms, an isolation window of 4 m / z, and stepped normalized collision energy (NCE) of 35, 50, and 75 eV in PRM mode. The tune file set a sheath gas flow rate of 50, an auxiliary gas flow rate of 15, a sweep gas flow rate of 1, a spray voltage of -2.75 kV, a capillary temperature of 35O°C, an S-lens RF level of 50.0, and an auxiliary gas temperature of 425°C. To quantify the concentrations of LAA, the raw spectrum of LAA was imported into the Thermo Xcalibur processing setup module to manually verify the retention times and m / z values in negative mode. The processing method was then employed for the initial automatic integration of the peak areas in the Quan Browser, followed by manual integration for both LAA standards and biological samples. The resulting peak area table was exported to generate a linear standard curve for LAA and interpolate the LAA concentration in biological samples.

[0281] Quantification of L-ascorbic acid using HPLC with coulometric electrochemical detection. Intracellular ascorbic acid was analyzed by HPLC with coulometric electrochemical detection as described, with minor modifications^?. HEK293 cells were seeded in 6-well plates and, upon adherence, were exposed to different concentrations of ascorbic acid for 24 hours. At the end of the treatment, cells were harvested and immediately placed on ice. Cells were centrifuged (500 xg for 10 minutes at 4°C) and washed twice with ice-cold 1X PBS, followed by the addition of 60% HPLC-grade methanol + 1 mM EDTA (250 pL of methanol-EDTA solution / lxio6cells) to the mixture. The mixture was then vortexed thoroughly and incubated on ice for 10 minutes. After incubation, the mixture was centrifuged at 18000 xg for 10 minutes at 4°C. The supernatant was collected and stored at -80 °C for further analysis by HPLC.

[0282] Chromatographic separation was performed using a C18 column (5pm, 4.6 mm 25cm ODS-DABS, Ultrasphere 240002; Beckman Coulter, Brea, CA, USA) with an HPLC system (Waters, Milford, MA, USA) and Coulochem III detector from ESA-Dionex (Chelmsford, MA, USA). Detector settings were electrode 2, 250 mV; electrode 1, 50 mV. Mobile phase was prepared with 0.05 M sodium phosphate monobasic, 0.05M sodium acetate anhydrous, 189 pM dodecyltrimethylammonium chloride (Sigma), and 36.6 pM tetraoctylammonium bromide (Santa Cruz Biotechnology, USA). Tetraoctylammonium bromide was dissolved in 100% methanol (HPLC-grade), andAttorney Docket No. 103362-088WO1 all other reagents were dissolved in HPLC-grade water (Milli-Q; Millipore). Finally, the methanol percentage was adjusted to 30% of the final volume, and pH was adjusted to 5.7 with orthophosphoric acid. The column was equilibrated with mobile phase at imL / min for 24-36 hours prior to running samples. Standards and samples were analyzed with mobile phase flow rate of lml / min and the injection volume was 10 pL.

[0283] Principles and Limitations of Biochemical AA Detection

[0284] One of the most common assays for AA detection is the plate-based biochemical fluorescence assay12. This assay is relatively simple and requires the lysis of a cell population. AA in the cell extract is first chemically oxidized into DHA using TEMPOL (FIG. 1A). The carbonyl groups on DHA then condense with the diamine group on o-phenylenediamine (OPDA), forming the fluorescent product OPDA-DHA that is detectable by a spectrometer (FIG. 1A). However, the plate-based assay can only measure the average AA level of a cell population, limiting its use in quantifying the AA concentration of individual cells.

[0285] To study AA in a cell population with heterogeneous AA concentrations, we aimed to develop an accessible, sensitive, and high-throughput method to detect AA at singlecell levels. Based on our understanding of the principle behind the biochemical assay, we hypothesized that a fluorescence compound with a diamine group could potentially be used for AA detection. After searching commercially available fluorescence compounds with a diamine group similar to OPDA, we identified 4,5-diaminofluorescein 2 (DAF-2, FIG. 1B), a common indicator for nitric oxide (NO). DAF-2 reacts with NO via N-nitrosation to form the fluorescent product DAF-2 triazole (DAF-2T) (FIG. 1C)22>23. Like OPDA, DAF-2 contains the diaminobenzene group (red circle, FIGs. 1A-1B), and it could potentially react with the carbonyl groups on DHA (FIG. 1D). Indeed, the reaction between DAF-2 and DHA has previously been reported to contribute to “non-specific” background during NO measurement by generating a highly fluorescent product DAF-2-DHA (FIG. 1D)21. We speculate that this “non-specific” reaction between DAF-2 and DHA could allow us to detect intracellular AA on the single cell level.

[0286] Development of Single-cell Ascorbate Level Sensing Assay (SALSA)Attorney Docket No. 103362-088WO1 To test if DAF-2 could be used to measure intracellular AA, we cultured HEK293T cells in the presence or absence of 1 mM L-ascorbic acid (AA) for 24 hours (FIG. 1E). To confirm AA uptake, intracellular AA concentration was quantified using the platebased fluorescence AA assay (FIG. 1A). Our data show that the intracellular AA levels were substantially increased only in the presence of AA (FIG. 1F), consistent with the notion that most tissue culture media is devoid of AA due to oxidation2^ To test if DAF-2 reacts with endogenous DHA in our system, cells cultured with or without AA were labeled with the cell -permeable DAF-2 diacetate (referred to as DAF-2 herein) and analyzed by FACS analysis. We found that the fluorescence signals of DAF-2 were similar regardless of intracellular AA levels (FIG. 1G), suggesting that DAF-2 does not react significantly with DHA derived from endogenous AA oxidation under these conditions. Inspired by the plate-based fluorescence AA assay, we hypothesized that oxidizing intracellular AA using TEMPOL could facilitate the detection of AA using DAF-2. TEMPOL, a cell-permeant compound, has been used in cell culture as a superoxide dismutase mimic and a radical scavenger, and it was shown to safely improve heart function and decrease lipid peroxidation in a mouse model2^26. Indeed, the DAF-2 fluorescence increased significantly upon TEMPOL treatment in AA- but not in the mock-treated cells (FIG. 1G), suggesting TEMPOL oxidized AA into DHA, which then reacted with DAF-2. These results show that the combination of DAF-2 and TEMPOL enables specific AA detection, a method we termed Singlecell Ascorbate Level Sensing Assay (SALSA; FIG. 1H).

[0287] To optimize SALSA, we tested multiple labeling conditions. We varied concentrations of DAF-2 from 1 to 20 pM (FIGs. 1I-1J), and all concentrations effectively distinguished between mock- and AA-treated cells (FIG. 11). Since 10 pM of DAF-2 significantly increased fluorescence intensity in the presence of AA, we used this concentration for subsequent experiments. We also tested DAF-FM (4-amino-5-methylamino-2',7'-difluorofluorescein, diacetate); however, due to its lower specificity for NO compared to DAF-2, it was less effective for AA detection (FIGs. 11-1J). Extending the DAF-2 labeling time from 10 to 20 minutes showed no significant difference (FIGs. 1K-1L), so we labeled the cells for 10 minutes in further experiments. Based on these results, we established the basic SALSA labeling protocol.

[0288] Unexpected Emission Spectrum Red-shift Significantly Enhanced SALSAAttorney Docket No. 103362-088WO1 On the flow cytometer, DAF-2 is usually excited using the 488 nm blue laser, and the emission is detected primarily with the FITC (fluorescein isothiocyanate) green channel (530 / 30 nm; FIG. 1H). Unexpectedly, we noticed that the DAF-2 fluorescent profile from mock- treated cells was not able to compensate for the spillover of DAF-2 signal from AA-treated cells, suggesting a qualitative difference between the two spectra (FIG.2A). Further analysis revealed that the AA-induced SALSA signal (DAF-2 and TEMPOL treatment) significantly spilled from FITC to the downstream channels, in particular the PE (phycoerythrin) red-orange channel (585 / 42 nm; FIGs. 2A-2C, 6A-6B). This observation indicates that the DAF-2-DHA emission spectrum is red-shifted compared to DAF-2 alone and DAF-2T. While not excited by the 637 nm red laser, DAF-2-DHA showed increased fluorescence in the BV510 channel when excited by the 405 nm violet laser (FIGs. 2C, 6A-6B). To ensure the results are reproducible across different cytometer setups, we observed similar outcomes when using another cytometer (BD FACSymphony; FIGs. 2D-2E, 6A-6B). Taking advantage of the differential spectra, we designated the FITC / BB515 green channel as SALSAVerdeand the following PE / BB630 red-orange channel as SALSAR°ia. We found that SALSAR°iais significantly more sensitive than SALSAVerdefor AA detection (FIGs. 2C, 2E, 6A-6B). However, as SALSAVerdeemits green fluorescence regardless of AA, it could be used to identify the successfully labeled cells with DAF-2. Depending on the equipment and experimental setup, the signal in AA-treated cells measured using SALSAR°ia(red-orange channel) can be at least 20 times higher than in mock-treated cells (FIG. 2E). This highlights the superior sensitivity of SALSAR°iacompared to SALSAVerdefor intracellular AA detection.

[0289] To assess whether SALSA is quantitative, we cultured HEK293T cells in varying concentrations of AA. The intracellular AA levels were evaluated using biochemical assay and SALSA (FIGs. 2F-2J). We found that the intracellular AA and SALSA signals are proportional to the supplemented AA (FIGs. 2F-2J). The SALSA signal has a strong linear relationship to the intracellular AA (FIGs. 2H and 2J), and SALSAR°jais about 10 times more potent than SALSAVerde(FIGs. 2J vs. 2H). Similar results were observed in another flow cytometer and cell type (FIGs. 7A-7F). Since the biochemical assay and SALSA utilize the same chemical reaction to detect AA, we independently validate the intracellular AA levels using high-performance liquid chromatography (HPLC) and liquid-chromatography mass spectrometry (LC-MS;Attorney Docket No. 103362-088WO1 FIGs. 7G-7I). Using these methods, we confirm the positive correlation between the supplemented and intracellular AA levels and TEMPOL-mediated AA oxidation (FIGs. 2I-2J). To further demonstrate the potential of SALSA for absolute quantification, we used streptolysin O to load the cells with various AA levels, followed by SALSA (FIG. 2K). Permeabilized cells loaded with AA showed a strong linear correlation between AA levels and SALSA signals (FIGs. 2L-2O). Thus, our results showed that SALSA is a quantitative method that can measure intracellular AA at a single-cell level in multiple cell types.

[0290] SALSA can Effectively Detect AA in the Presence of NO

[0291] DAF-2 is primarily an indicator dye for NO detection in biological systems. To assess the effect of NO on SALSA, we used a murine macrophage cell line, Raw264.7, which produces NO after lipopolysaccharide (LPS) and interferon-g (IFNg) stimulation. Twenty-four hours after stimulation, Raw264.7 cells significantly increased in size, upregulated the expression of an activation marker CD80, induced nitric oxide synthetase (iNOS; FIG. 3A), and increased NO production (FIG. 3B). The addition of AA had no significant effect on the levels of CD80, iNOS, and NO (FIGs. 3A-3B). While both SALSAVerde(BB515 channel) and SALSAR°ia(BB630 channel) differentiated the mock- and AA-treated resting cells (FIGs. 3C-3E), SALSAR°iaprovides a superior AA-to-mock ratio (23.7X background) than SALSAVerde(3.3X background). However, while SALSAVerdebecame ineffective for stimulated NO-producing cells, SALSAR°iacould still discern the mock and AA groups (6.7X background; FIGs. 3C-3E). Similar results were obtained when cells were cultured with lower, more physiological AA concentrations (FIGs. 8A-hJ). We found that despite the presence of NO, SALSA can distinguish the intracellular AA in cells cultured with physiological levels of AA (FIGs. 8A-hJ). The interference from NO could be further diminished by using the iNOS inhibitor 1400W (FIGs. 8A-8H) and using the SALSAR°iachannel, which is more specific for the detection of AA (FIGs.

[0292] 8D, 8F, 8H). Therefore, our results demonstrated SALSA’s effectiveness in AA detection despite NO.

[0293] Reducing NO Interference Further Augmented SALSA Specificity

[0294] To test whether NO inhibition could improve SALSA’s AA detection, we stimulated Raw264.7 cells in the presence of 1400W, a specific iNOS inhibitor2?. The addition ofAttorney Docket No. 103362-088WO1 1400W abolished NO (FIG. 3B) and decreased the baseline SALSAVerdesignal in mock-treated cells, restoring the detection power to that observed in resting cells (FIGs. 3D, 3G). iNOS inhibition did not affect the SALSAR°iasignals (FIGs. 3F, 3H), supporting the notion that the SALSAR°iachannel is less susceptible to NO interference and is more specific for AA detection. To test whether quenching existing NO could improve the robustness of SALSA, we incubated the cells with the NO scavenger carboxy-PTIO for 30 minutes before SALSA. Like iNOS inhibition, NO quenching significantly improved SALSA detection (FIG.3I). Our results suggest that SALSA’s specificity for AA can be further augmented by removing NO in the samples.

[0295] Using SALSA to Assess Intracellular AA in a Heterogenous Population The major advantage of SALSA over plate-based biochemical assays is its ability to detect intracellular AA at a single-cell level instead of measuring the average AA levels in a cell population. Therefore, SALSA is well-suited for experiments that require the isolation of cells according to the intracellular AA levels, such as pooled CRISPR screens. To exemplify this idea, we used SALSA to assess the transporter involved in AA uptake in HEK293T cells. Two major AA transporters in mammalian cells responsible for AA uptake are SVCT1 (sodium-coupled vitamin C transporter 1, encoded by SLC23A1) and SVCT2 (SLC23A2). Using the lentiviral CRISPR system, we designed three single-guide RNAs (sgRNAs) to target each transporter28-2?. HEK293T cells were transduced with lentivirus expressing Cas9 and sgRNA, followed by puromycin selection to select transduced cells. CRISPR targeting efficiency was confirmed using TIDE assay, which quantifies the mutation frequency in a population by deconvoluting the traces from Sanger sequencing (FIG. 4A)3°. To recapitulate a pooled CRISPR screen scenario, WT, and CRISPR-targeted cells were cultured with AA for 24 hours, barcoded with antibodies, mixed, and analyzed using SALSA (FIGs. 4B-4D). Our data showed that barcoding with antibodies for a ubiquitous antigen f2M allowed us to identify the two mixed populations (FIG. 4C). While vector control (Ctrl) and targeting SVCTi had a neglectable effect on AA uptake (FIG. 4D-4E), SVCT2 mutation substantially decreased the AA uptake (FIG. 4D-4E). Our result is consistent with the RNA sequencing data from the Human Protein Atlas, which shows that the SLC23A2 expression is 15 ox higher than that of SLC23A131. In conclusion, our results demonstrate that SALSA is an accessible and simple method to measure AA at single-cell levels specifically.Attorney Docket No. 103362-088WO1 SALSA Reveals Differential Intracellular AA Among Immune Cells Ex vivo

[0296] Having established the feasibility of SALSA in analyzing heterogeneous populations, we applied SALSA to investigate the intracellular AA in immune cells. To confirm the detected signals were bona fide, we used cells from Gulo-deficient (KO) mice, which cannot synthesize AA due to the mutation of L-gulonolactone oxidases2. Gulo-KO mice were either supplemented with (AA-sufficient) or maintained without AA (AA-deficient) for 3 weeks (FIG. 5A) when the plasma and intracellular levels of AA decreased significantly (FIGs. 9A-9B). To demonstrate the capability of SALSA, we established a flow cytometry staining panel with an additional 12 colors. Cells from blood, spleens, and lymph nodes were isolated from the two groups of Gulo-KO mice and analyzed using the multicolor panel (FIGs. 5B-5D). In the blood of control mice, B cells (CD19+) and myeloid cells (CDnb+) have higher AA compared to CD4 and CD8 T cells (FIG. 5B), consistent with earlier findings in humans33. While no significant differences in cell number were observed after AA depletion (FIG. 5B), all cell types in the blood and spleen exhibited decreased SALSA signal, consistent with the decrease in intracellular AA and validating SALSA’s specificity (FIGs. 5C-5D). The AA level in CD4+T cells was relatively sensitive to AA depletion. Their signal decreased to almost baseline in AA-deficient mice (FIG. 5C). Interestingly, blood CD8+T cells retained relatively more AA after AA depletion when compared to other cell types (FIG. 5C), suggesting CD8+T cells may either metabolize less AA or have higher AAuptake / ret ention. In the spleen, while myeloid and B cells maintained high AA levels similar to their counterparts in the blood, CD4 and CD8 T cell subsets had markedly decreased AA levels (FIGs. 5C-5D). Since the SALSA signal was widely distributed in CD4 and CD8 T cells, we hypothesize that T cell subsets may have different AA levels. Indeed, using CD44 as a memory / activation marker, we found that CD44hiT cells have higher AA than naive T cells (FIG. 5D). This data suggests that memory T cells may require more AA to regulate their self-renewal potential, similar to a recent study of AA in hematopoietic stem cells34.

[0297] The three-week AA depletion also decreased intracellular AA concentrations in the thymus, the primary lymphoid organ where T cells develop (FIG. 5E). Thymocytes were gated into different stages of development based on their expression of CD4 and CD8, namely CD4“CD8“ double negative (DN), CD4+CD8+double positive (DP),Attorney Docket No. 103362-088WO1 CD4+CD8“ single positive (CD4SP), and CD4~CD8+single positive (CD8SP; FIG.5F). Double negative thymocytes (DN) were subdivided into four maturation stages (DN1-4). SALSA revealed that the intracellular AA levels primarily declined with maturation, following the order from DN > DP > CD4 / 8SP (FIG 5G). Among all major thymic subsets, we found that AA depletion preferentially increased the frequency of CD4SP (FIGs. 5H, 5J). We further divided the CD4SP subset based on the expression of CD 25, a marker for thymic regulatory T cells and precursorsss, and CD24, a maturation marker inversely correlated with thymocyte maturations6. We found a slightly higher percentage of immature CD24hicells, suggesting a potential minor maturation impairment (FIG. 5K). Consistent with the inverse relationship between AA levels and maturation states, the immature CD24hicells have higher AA than CD2410cells (FIG. 5G). Interestingly, the CD25+regulatory T cells or precursors have higher AA than other CD4SP at the same maturation stage (FIG.5G), suggesting a potential function of AA in natural regulatory T cells. Thus, the data indicate that AAmaybe differentially required throughout thymocyte development. Furthermore, our results demonstrate SALSA’s sensitivity and its ability to discern AA levels within a heterogeneous cell population, which may illuminate AA’s potential cellular functions.

[0298] DISCUSSION

[0299] During our studies of AA in the epigenetic regulation of immune cells, we realized that a limitation to understanding its cellular function was that no simple method exists to measure intracellular AA levels in individual cells. We rationalized that DAF-2, a common nitric oxide (NO) probe with a similar diamine group as o-phenylenediamine, might also react with dehydroascorbate (DHA), a concept previously observed as an artifact in NO studies but not harnessed for ascorbate detection21. In principle, the SALSA signal represents the combined level of ascorbate and a minor level of DHA, which is relatively unstable and at a low level in the cells. However, our data showed no significant increase in DAF-2 fluorescence in mock-versus AA-treated cells (FIG. 1G), suggesting the reaction between endogenously occurring DHA and DAF-2 was insignificant under our conditions. Upon TEMPOL-mediated AA oxidation, DAF-2 green fluorescence (SALSAVerde) noticeably increased, potentially via a mechanism similar to that of the biochemical assay (FIG. 1A). WhileAttorney Docket No. 103362-088WO1 promising, the signal-to-background ratio for SALSAVerdesignal was not as robust as expected.

[0300] During our analysis of NO-producing macrophages, we serendipitously found that the spectrum of DAF-2-DHA differs from those of DAF-2 and DAF-2T. Multiparameter flow cytometry analyses revealed that the DAF-2-DHA has a significantly higher emission in the channels following SALSAVerde(FITC / BB515; FIGs. 2C, 2E). As the signals peaked in the red-orange PE / BB630 channels, we designated them the SALSAR°iachannel. Compared to SALSAVerde, SALSAR°iahad a superior signal-to-background ratio (FIGs. 2G-2J) and was more resistant to signal interference from NO (FIGs. 3A-3H). SALSA’s multiple detection channels are advantageous. For instance, due to the basal fluorescence of DAF-2, the SALSAVerdecan first be used to identify the successfully labeled cells. SALSAR°iacan subsequently detect AA with greater specificity. Our observed red-shifted spectrum differs from the findings of a previous study, which suggested similar spectra for DAF-2 derivatives21. The discrepancy could be due to the methodology (spectral scanning v. flow cytometry) and analysis conditions (purified compounds v. whole cells). Besides SALSAR°ia, our analysis also revealed additional spectral changes in the channels excited by UV (355 nm) and violet (405 nm) lasers (FIG. 2E), further underscoring the unique spectral profile of DAF-2-DHA.

[0301] Given the reactivity between DAF-2 and NO, one potential limitation of SALSA is the interference from NO. Indeed, the presence of NO significantly masked the signal on SALSAVerde(FIG. 3D). To alleviate this issue, we found that SALSAR°iasignal is substantially less affected by NO (FIG. 3E), an effect attributed to the potential spectral differences between DAF-2-DHA and DAF-2T. Importantly, we showed that iNOS inhibition or NO quenching could negate the interference and decrease the background fluorescence (FIGs. 3G-3I). NO quenching may be more practical as it can be used immediately before SALSA and lower the potential impact on the cells’ behavior due to the prolonged iNOS inhibition (24 hours in our experiments). While stimulated cells may have increased background for SALSA independent of NO (FIGs. 3H-I), combining these mitigating approaches significantly increased the signal-to-background ratio. With further optimization, the ability to modulate NO and measure AA may allow for a better understanding of NO-enzymology, where ascorbate plays a role as a co-factor37,38.Attorney Docket No. 103362-088WO1 SALSA offers several key advantages over existing methods for ascorbate detection. It is readily accessible, relying on commercially available, cell-permeable reagents— DAF-2-AM and TEMPOL— that exhibit low cytotoxicity. DAF-2 is widely used as a nitric oxide (NO) probe, while TEMPOL functions as a superoxide dismutase (SOD) mimic and radical scavenger. The assay has a low technical barrier, requiring only a standard flow cytometer equipped with a 488 nm laser. Despite its simplicity, SALSA is robust and scalable, enabling the single-cell analysis of AA across millions of cells. The dual fluorescence channels— SALSAVerdeand SALSAR°ia— along with TEMPOL-mediated oxidation provide a means to normalize across cell types and account for variability in DAF-2 labeling. In addition to AA, SALSA can potentially be used to analyze the dynamics of DHA with minor modifications by omitting TEMPOL. In fact, we routinely include the “no TEMPOL” control to determine the SALSA signal attributable to DHA and AA. Our data show that SALSA can be combined with immunophenotyping markers to resolve AA heterogeneity across cell populations (FIGs. 4A-4E, 5A-5K), making it well-suited for high-throughput functional genomics and immune profiling. Importantly, with appropriate calibration and optimization, SALSA enables not only relative quantification but also accurate estimates of intracellular AA content per cell (FIGs. 2K-2O). Coupled with fluorescence barcodings?*, SALSA can accommodate tens to hundreds of samples in a single experiment, further enhancing its throughput and utility.

[0302] While promising, SALSA has several limitations. DAF-2 is relatively susceptible to photobleaching4°, requiring careful handling to maintain signal integrity. Cells should be analyzed as soon as possible, as we found that non-specific signals increase after 1-2 hours post-SALSA labeling. The quality of the reagents is essential for the success of SALSA, as the performance of DAF-2 significantly declines after repeated freeze-thaw cycles. Importantly, since the SALSA signal is detected in multiple channels across lasers, users must be cautious with flow cytometry panel designs and compensation controls to avoid misinterpreting results. Additionally, the current SALSA workflow necessitates live cells and is incompatible with fixation. This issue can be circumvented by sorting cells based on SALSA signals before intracellular staining.

[0303] Beyond its utility as a research tool, SALSA holds promise as a biomarker. Plasma AA levels fluctuate due to diet"*1, thus limiting their value as indicators of physiologicalAttorney Docket No. 103362-088WO1 status. In contrast, intracellular AA concentrations vary by cell type and may reflect a more stable, biologically relevant pool of AA. Our data show that immune cells possess heterogeneous AA levels (FIGs. 5A-5K), with different subsets exhibiting variable susceptibility to AA depletion. This cell type-specific variability could provide a composite snapshot of an individual's recent vitamin C status. Such an approach may be especially informative in infection, chronic inflammation, or oxidative stress, where AA turnover is elevated. Epidemiologic studies have associated decreased plasma AA levels in humans with elevated risk of certain cancers and chronic diseases, particularly cardiovascular disease's, in these cases, with the understanding of the dynamics and differential AA metabolism in different immune cells, SALSA could potentially determine the recent AA status in an individual. It has the potential to be used to classify disease states, analogous to how hemoglobin AiC and glucose measurements have complementary value in diabetes management44. Finally, SALSA can be used more broadly to study AA’s crucial role in redox biology. As an antioxidant, AA modulates reactive oxygen species levels alone and in combination with glutathione10. SALSA may be used to investigate the dynamics between various classes of antioxidants in individual cells.

[0304] In conclusion, SALSA is a sensitive and accessible method for detecting intracellular ascorbate at single-cell resolution. By leveraging a repurposed NO probe and revealing a unique, red-shifted fluorescence signature of the DAF-2-DHA reaction, SALSA fills a longstanding methodological gap in vitamin C research. Its compatibility with high-throughput flow cytometry, immunophenotyping, and functional genomics makes it a versatile tool for probing AA biology in diverse cell types and disease contexts. A longstanding goal of ascorbic acid studies, and more broadly, nutrition research, is to characterize how nutrient concentrations regulate function45. As a single-cell assay, SALSA is a key tool that unlocks future investigations in nutrition, redox biology, and metabolic dynamics in immune cells. The references cited herein and below are hereby incorporated by reference to disclose and describe the methods or materials in connection with which the publications are cited or to provide background for the present disclosure. Any incorporation by reference of documents herein and below is limited such that no subject matter is incorporated by reference that is contrary to the explicit disclosure herein. In the event of inconsistent usages between this document and thoseAttorney Docket No. 103362-088WO1 documents so incorporated by reference herein and below, the use in the incorporated references should be considered supplementary to that of this document; for irreconcilable inconsistencies, the usage in this document controls.

[0305] 1. Dresen E, Lee ZY, Hill A, Notz Q, Patel JJ, Stoppe C. History of scurvy and use of vitamin C in critical illness: A narrative review. Nutr Clin Pract. 2O23;38(I):46-54.

[0306] 2. Nishikimi M, Yagi K. Molecular basis for the deficiency in humans of gulonolactone oxidase, a key enzyme for ascorbic acid biosynthesis. Am J Clin Nutr.

[0307] 1991;54(6 Suppl):i2O3S-i2o8S.

[0308] 3. Savini I, Rossi A, Pierro C, Avigliano L, Catani MV. SVCT1 and SVCT2: key proteins for vitamin C uptake. Amino Acids. 2OO8;34(3):347-355.

[0309] 4. Evans RM, Currie L, Campbell A. The distribution of ascorbic acid between various cellular components of blood, in normal individuals, and its relation to the plasma concentration. Br J Nutr. i982;47(3):473-482.

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[0338] 33. Bergsten P, Amitai G, Kehrl J, Dhariwal KR, Klein HG, Levine M. Millimolar concentrations of ascorbic acid in purified human mononuclear leukocytes. Depletion and reaccumulation. The Journal of biological chemistry. i99O;265(5):2584-2587.Attorney Docket No. 103362-088WO1 34. Comazzetto S, Cassidy DL, DeVilbiss AW, et al. Ascorbate deficiency increases quiescence and self-renewal in hematopoietic stem cells and multipotent progenitors. Blood. 2O25;145(1):114-126.

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[0350] 43. Block G. Vitamin C and cancer prevention: the epidemiologic evidence. Am J Clin Nutr. 1991553(1 Suppl):27oS-282S.Attorney Docket No. 103362-088WO1 44. Ebenuwa I, Violet PC, Padayatty S, et al. Abnormal urinary loss of vitamin C in diabetes: prevalence and clinical characteristics of a vitamin C renal leak. Am J Clin Nutr. 2O22; II6(I):274-284.

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[0354] The compositions and methods of the appended claims are not limited in scope by the specific compositions and methods described herein, which are intended as illustrations of a few aspects of the claims, and any compositions and methods that are functionally equivalent are intended to fall within the scope of the claims. Various modifications of the compositions and methods, in addition to those shown and described herein, are intended to fall within the scope of the appended claims. Further, while only certain representative compositions and method steps disclosed herein are specifically described, other combinations of the compositions and method steps also are intended to fall within the scope of the appended claims, even if not specifically recited. Thus, a combination of steps, elements, components, or constituents may be explicitly mentioned herein; however, other combinations of steps, elements, components, and constituents are included, even though not explicitly stated.

Claims

Attorney Docket No. 103362-088WO1WHAT IS CLAIMED IS:

1. A method for detecting ascorbate within a cell comprising:contacting the cell with a compound of Formula (I) or Formula (II):wherein:R1aand R1bare independently selected from -OR4 and -NR5aR5b;R2aand R2bare independently selected from hydrogen and halo;R3 is -NHR6;R4 is independently selected at each occurrence from hydrogen and -C(O)R7;R5aand R5bare independently selected at each occurrence from hydrogen and C1-C6 alkyl;R6is selected from hydrogen and C1-C6 alkyl; andR is selected from C1-C6 alkyl and phenyl;-contacting the cell with an oxidant capable of oxidizing ascorbate into dehydroascorbic acid;-irradiating the cell with light having a wavelength from about 400 nm to about 500 nm; andAttorney Docket No. 103362-088WO1-detecting fluorescence from the cell having a wavelength from about 500 nm to about 650 nm, whereupon detection of the fluorescence confirms the presence of ascorbate within the cell.

2. The method of claim 1, wherein the compound of Formula (I) is administered as an isomeric form of Formula (I’):R1aO® R1b^\R2boNH2(I’).

3. The method of claim 1, wherein the compound of Formula (II) is administered as an isomeric form of Formula (II’):R1aR2i^\R2bO(II’).

4. The method of any one of claims 1-3, wherein Rlaand Rlbare each -OH.

5. The method of any one of claims 1-3, wherein Rlaand Rlbare each -OC(O)CH3.

6. The method of any one of claims 1-3, wherein Rlaand Rlbare each -N(CH3)2.

7. The method of any one of claims 1-3, wherein Rlaand Rlbare each -N(CH2CH3)2.

8. The method of any one of claims 1-7, wherein R2aand R2bare each hydrogen.

9. The method of any one of claims 1-7, wherein R2aand R2bare each fluoro.

10. The method of any one of claims 1-7, wherein R2aand R2bare each chloro.

11. The method of any one of claims 1-10, wherein Rs is -NH2.Attorney Docket No. 103362-088WO112. The method of any one of claims 1-10, R3 is -NH(CI-C6 alkyl).

13. The method of any one of claims 1-10, wherein Rs is -NH(CH3).

14. The method of claim 1, wherein the compound of Formula (I) or Formula (II) is selected from:(DAF-FM DA),(DAF-2),(DAF-2 DA),(DAF-4),Attorney Docket No. 103362-088WO1(DAR-4),(DAR-4M), andAttorney Docket No. 103362-088WO1(DAR-M).15- The method of claim 1, wherein the compound of Formula (I) or Formula (II)(DAF-2).

16. The method of any one of claims 1-15, wherein the oxidant comprises a nitroxide.

17. The method of any one of claims 1-16, wherein the oxidant is selected from (4-hydroxy-2,2,6,6-tetramethylpiperidin-i-yl)oxyl (TEMPOL) or 3-Carboxy-2, 2,5,5-tetramethyl-i-pyrrolidinyloxy, free radical (3-Carboxy-PROXYL).

18. The method of any one of claims 1-17, wherein the oxidant is (4-hydroxy-2,2,6,6-tetramethylpiperidin-i-yl)oxyl (TEMPOL).

19. The method of any one of claims 1-18, wherein contacting the cell with the compound of Formula (I) or Formula (II) comprises contacting the cell with a solution of the compound of Formula (I).

20. The method of claim 19, wherein the compound of Formula (I) or Formula (II) has a concentration in the solution from about 1 micromolar to 20 micromolar.

21. The method of claim 19 or claim 20, wherein the compound of Formula (I) or Formula (II) has a concentration in the solution of about 10 micromolar.

22. The method of any one of claims 1-21, wherein contacting the cell with the oxidant comprises contacting the cell with a solution of the oxidant.Attorney Docket No. 103362-088WO123. The method of claim 22, wherein the oxidant has a concentration in the solution from about 1 millimolar to about 50 millimolar.

24. The method of claim 22 or claim 23, wherein the oxidant has a concentration in the solution of about 20 millimolar.

25. The method of any one of claims 1-24, wherein the cell is irradiated with a light source.

26. The method of claim 25, wherein the light source comprises a laser.

27. The method of any one of claims 1-26, wherein the cell is irradiated with light having a wavelength of 488 nm.

28. The method of any one of claims 1-27, wherein irradiating the cell and / or detecting the fluorescence occurs via flow cytometry.

29. The method of any one of claims 1-27, wherein irradiating the cell and / or detecting the fluorescence occurs via fluorescence microscopy.

30. The method of any one of claims 1-29, further comprising contacting the cell with a nitric oxide synthase inhibitor or nitric oxide quencher prior to irradiating the cell with light.

31. The method of any one of claims 1-30, wherein the method detects a concentration of ascorbate within the cell.

32. The method of claim 31, wherein a median fluorescent intensity (MFI) of the fluorescence is correlated to the concentration of ascorbate in the cell.

33. The method of any one of claims 1-32, wherein the cell is a mammalian cell.

34. The method of any one of claims 1-33, wherein the cell is a human cell.

35. The method of any one of claims 1-34, wherein the cell is associated with a disease or disorder.

36. The method of claim 35, wherein the disease or disorder comprises a hematological malignancy, a metabolic disorder, or an immunodeficiency.

37. The method of claim 35 or 36, wherein the disease or disorder is associated with a decreased concentration of ascorbate within the cell compared to a healthy cell.Attorney Docket No. 103362-088WO138. The method of claim 35 or 36, wherein the disease or disorder is associated with an increased concentration of ascorbate within the cell compared to a healthy cell.

39. The method of any one of claims 1-38, wherein the cell is a blood cell.

40. The method of any one of claims 1-38, wherein the cell is an immune cell.

41. The method of any one of claims 1-38, wherein the cell is a cancer cell.

42. The method of claim 41, wherein the cancer cell is a leukemia cell.

43. The method of any one of claims 1-42, wherein the cell is provided in a sample from a subject.

44. The method of claim 43, wherein the sample is a blood sample, a bone marrow sample, a tumor sample, a tonsil sample, a spleen sample, or a lymph node sample.

45. The method of claim 43 or 44, wherein the subject is a human.

46. A kit comprising:a compound of Formula (I) or Formula (II):wherein:Rlaand Rlbare independently selected from -OR4 and -NRsaRsb;R2aand R2bare independently selected from hydrogen and halo;R3 is -NHR6;Attorney Docket No. 103362-088WO1R4 is independently selected at each occurrence from hydrogen and -C(O)R?;Rsaand R5bare independently selected at each occurrence from hydrogen and Ci-Ce alkyl;R6is selected from hydrogen and Ci-Ce alkyl; andR is selected from Ci-Ce alkyl and phenyl; andan oxidant capable of oxidizing ascorbate into dehydroascorbic acid.

47. The kit of claim 46, wherein the compound of Formula (I) is provided as an isomeric form of Formula (I’):

48. The kit of claim 46, wherein the compound of Formula (II) is provided as an isomeric form of Formula (II’):

49. The kit of any one of claims 46-48, wherein Rlaand Rlbare each -OH.

50. The kit of any one of claims 46-48, wherein Rlaand Rlbare each -OC(O)CH3.

51. The kit of any one of claims 46-48, wherein Rlaand Rlbare each -N(CH3)2.

52. The kit of any one of claims 46-48, wherein Rlaand Rlbare each -N(CH2CH3)2.

53. The kit of any one of claims 46-52, wherein R2aand R2bare each hydrogen.

54. The kit of any one of claims 46-52, wherein R2aand R2bare each fluoro.Attorney Docket No. 103362-088WO155- The kit of any one of claims 46-52, wherein R2aand R2bare each chloro.

56. The kit of any one of claims 46-55, wherein Rs is -NH2.

57. The kit of any one of claims 46-55, Rs is -NH(CI-C6 alkyl).

58. The kit of any one of claims 46-55, wherein Rs is -NH(CH3).

59. The kit of claim 46, wherein the compound of Formula (I) or Formula (II) is selected from:(DAF-FM DA),(DAF-2),(DAF-2 DA),Attorney Docket No. 103362-088WO1(DAF-4 DA),(DAR-2),(DAR-4),Attorney Docket No. 103362-088WO1(DAR-4M), and(DAR-M).6o. The kit of claim 46, wherein the compound of Formula (I) or Formula (II) is(DAF-2).

61. The kit of any one of claims 46-60, wherein the oxidant comprises a nitroxide.

62. The kit of any one of claims 46-61, wherein the oxidant is selected from (4-hydroxy-2,2,6,6-tetramethylpiperidin-i-yl)oxyl (TEMPOL) or 3-Carboxy-2, 2,5,5-tetramethyl-i-pyrrolidinyloxy, free radical (3-Carboxy-PROXYL).

63. The kit of any one of claims 46-62, wherein the oxidant is (4-hydroxy-2, 2,6,6-tetramethylpiperidin-i-yl)oxyl (TEMPOL).

64. The kit of any one of claims 46-63, further comprising a nitric oxide synthase inhibitor or nitric oxide quencher.Attorney Docket No. 103362-088WO165. The kit of any one of claims 46-64, wherein the compound of Formula (I) or Formula (II) is provided as a solution of the compound of Formula (I).

66. The kit of claim 65, wherein the compound of Formula (I) or Formula (II) has a concentration in the solution from about 1 micromolar to 20 micromolar.

67. The kit of claim 65 or claim 66, wherein the compound of Formula (I) or Formula (II) has a concentration in the solution of about 10 micromolar.

68. The kit of any one of claims 46-67, wherein the oxidant is provided as a solution of the oxidant.

69. The kit of claim 68, wherein the oxidant has a concentration in the solution from about 1 millimolar to about 50 millimolar.

70. The kit of claim 68 or claim 69, wherein the oxidant has a concentration in the solution of about 20 millimolar.

71. A method of measuring a concentration of ascorbate in a cell comprising:-contacting the cell with a compound of Formula (I) or Formula (II):wherein:Rlaand Rlbare independently selected from -OR4 and -NRsaRsb;R2aand R2bare independently selected from hydrogen and halo;R3 is -NHR6;R4 is independently selected at each occurrence from hydrogen and -C(O)R?;Attorney Docket No. 103362-088WO1R5aand R5bare independently selected at each occurrence from hydrogen and Ci-Ce alkyl;R6is selected from hydrogen and Ci-Ce alkyl; andR is selected from Ci-Ce alkyl and phenyl;-contacting the cell with an oxidant capable of oxidizing ascorbate into dehydroascorbic acid;-irradiating the cell with light having a wavelength from about 400 nm to about 500 nm; and-measuring a median fluorescent intensity (MFI) of fluorescence from the cell, wherein the fluorescence has a wavelength from about 500 nm to about 650 nm; wherein the MFI is correlated to the concentration of ascorbate in the cell.

72. A method of diagnosing or assessing a disease or disorder within a subject associated with a cell, where the disease or disorder is associated with an alteration of a concentration of ascorbate within the cell compared to a healthy cell, the method comprising:-contacting the cell with a compound of Formula (I) or Formula (II):wherein:Rlaand Rlbare independently selected from -OR4 and -NRr>aR5b;R2aand R2bare independently selected from hydrogen and halo;Attorney Docket No. 103362-088WO1R3 is -NHR6;R4 is independently selected at each occurrence from hydrogen and -C(O)R?;Rsaand R5bare independently selected at each occurrence from hydrogen and Ci-Ce alkyl;R6is selected from hydrogen and Ci-Ce alkyl; andR is selected from Ci-Ce alkyl and phenyl;-contacting the cell with an oxidant capable of oxidizing ascorbate into dehydroascorbic acid;-irradiating the cell with light having a wavelength from about 400 nm to about 500 nm; and-measuring a median fluorescent intensity (MFI) of fluorescence from the cell, wherein the fluorescence has a wavelength from about 500 nm to about 650 nm; wherein the MFI is correlated to the concentration of ascorbate in the cell, and wherein a change in the concentration of ascorbate in the cell relative to a healthy cell is indicative of the presence or state of the disease or disorder.

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