Method for detecting lactic acid secretion by cells

Abstract

The present invention relates to a method for detecting single cell lactic acid secretion, and specifically provides a method for detecting cell lactic acid secretion, comprising: 1) incubating a fluorescent probe and cells in separate partitions, and 2) detecting the fluorescent signal, wherein the fluorescent signal of the partition where the lactic acid secreting cell is located is weakened. The method of the present application achieves accurate detection of cell lactic acid secretion.

Description

Technical Field

[0001] This invention relates to the field of biology, specifically to a method for detecting lactic acid secretion in cells. Background Technology

[0002] Because cells are heterogeneous, assessing cell behavior and activity at the single-cell level is crucial for understanding cell biology. High-throughput identification of high- and low-lactate-secreting cells at single-cell resolution is currently a research focus.

[0003] Lactic acid is a secreted substance, therefore, the detection of lactate in single cells needs to be performed in a microchamber (with isolation). For example, Weifei Zhang et al. (Anal. Chem. 2021, 93, 18, 6955–6960) used inkjet printing to encapsulate single A549 cells into droplets on a glass substrate, and then used mass spectrometry to detect lactate secretion in single cells. Amy Mongersun et al. (Anal. Chem. 2016, 88, 6, 3257–3263) anchored droplets containing single cells and lactate detection reagents (kits) of 0.5–1.2 nL in microwells, and observed the lactate secretion of cells in the droplets in real time using fluorescence microscopy. High-throughput detection of high / low lactate-secreting cells in microchambers has placed new demands on lactate detection methods. First, the microchambers are small in volume, and a small amount of lactic acid can cause a significant increase in the concentration in the system, thus requiring the detection method to have a sufficiently high detection threshold. Second, the microchambers are closed, making them incompatible with multi-step sample addition and dilution operations. Finally, a sufficient number of cells to be tested is required for statistical significance, thus requiring the method to have tens of thousands or even hundreds of thousands of microchambers for high-throughput and rapid detection.

[0004] Existing methods for lactate detection fall into two main categories: chemical analysis and enzymatic methods. Chemical analysis methods primarily include optical rotation, titration, and chromatography, utilizing the unique properties of lactate such as optical rotation, acidity, solubility, adsorption, and boiling point for quantification. However, these methods suffer from low automation and long detection times, thus failing to meet the demands of high-throughput detection. The most commonly used enzymes in enzymatic methods are lactate dehydrogenase and lactate oxidase, which obtain lactate concentration information by detecting the absorbance or fluorescence signal of the reaction products. However, methods for measuring absorbance (including lactate dehydrogenase and lactate hydrogenase) involve sample dilution and transfer, which contradicts the sealing requirements of microchambers and is therefore unusable. Fluorescent substances detected by enzymatic methods fall into two categories: NADH and fluorescent products obtained from NADH-oxidized fluorescent probes. The "fluorescent method for detecting NADH" requires the introduction of large amounts of NAD+, which affects cellular lactate secretion, when detecting high concentrations of lactate, thus failing to indicate the true level of cellular lactate secretion. When detecting high concentrations of lactic acid, the fluorescent probe may undergo excessive oxidation, leading to a decrease in the fluorescence signal of the fluorescent product and causing misjudgment.

[0005] As mentioned above, due to the extremely small size of the microchambers, the concentration of trace amounts of lactic acid secretion within them remains higher than the high-value quantitative detection of existing kits. The sealed nature of the microchambers prevents them from being detected after multiple dilutions as in conventional methods. This leads to a significant decrease in the detection signal due to excessive oxidation of these high lactic acid concentrations, to the point where they become indistinguishable from those in low-concentration lactic acid chambers, causing misdiagnosis. To avoid this problem, existing methods require the detection of a large number of cells (millions) within a very short period (a few minutes) initially when cells are encapsulated in the droplet (when lactic acid levels are low). However, the separation and detection of millions of cells in a microchamber takes several minutes or even tens of minutes, resulting in significant differences in the time point at which the signal is detected in each microchamber. Cells that were originally high in lactic acid secretion may become negative cells with low or no lactic acid secretion, making it impossible to determine the level of cellular lactic acid secretion based on the level of the lactic acid signal.

[0006] Existing methods struggle to detect lactate secreted by single cells. Detection of lactate secreted by a single cell requires detection within extremely small microcompartments. In qualitative analysis of high / low lactate-secreting cells within these microcompartments, traditional methods are time-consuming, difficult to differentiate between positive and negative signals, and thus limit throughput. Colorimetric methods are also challenging to perform in microcompartments due to optical path limitations, while fluorescence methods, which enable in-situ detection within microcompartments, have limitations in their detection range. Summary of the Invention

[0007] This invention proposes a qualitative method for lactate identification based on fluorescent probe consumption, used for high-throughput determination of high / low lactate-secreting cells in microcompartments. In the presence of high concentrations of lactate (> the detection upper limit given by the lactate kit), the fluorescent probe is completely / over-oxidized, and its fluorescence signal disappears completely; while in the presence of low concentrations of lactate, only a portion of the fluorescent probe is utilized, and its fluorescence signal is partially retained. Since the presence of lactate irreversibly reduces the fluorescence signal, eventually approaching zero, the requirements for differences in detection time points between different microcompartments are lower, enabling high-throughput determination of high / low lactate-secreting cells.

[0008] This invention provides a method for detecting cellular lactate secretion, comprising:

[0009] 1) Incubate fluorescent probes and cells in separate compartments.

[0010] 2) Detect the fluorescence signal at the first emission wavelength, wherein the fluorescence signal is weakened in the region where the lactate-secreting cells are located, and the first emission wavelength is 610nm-700nm.

[0011] In one or more embodiments, the fluorescent probe emits a reduced fluorescence signal at a first emission wavelength upon contact with lactic acid. Preferably, the fluorescent probe emits fluorescence at the first emission wavelength when not in contact with lactic acid, and the fluorescence at the first emission wavelength decreases upon contact with lactic acid.

[0012] In one or more embodiments, the first emission wavelength is 620-690 nm, more preferably 630-670 nm.

[0013] In one or more embodiments, the emission wavelength is the emission wavelength of the CY5 channel.

[0014] In one or more embodiments, the fluorescent probe exhibits enhanced fluorescence signal at a second emission wavelength upon contact with lactic acid. Preferably, the fluorescent probe exhibits weak fluorescence or essentially no fluorescence at the second emission wavelength when not in contact with lactic acid, and its fluorescence at the second emission wavelength is enhanced upon contact with lactic acid, wherein the second emission wavelength is 565 nm-605 nm.

[0015] In one or more embodiments, the fluorescent probe is over-oxidized at lactic acid concentrations greater than 30 μM, preferably greater than 50 μM, greater than 80, or greater than 100 μM.

[0016] In one or more embodiments, the individual partition further comprises one, more, or all of NAD, an enzyme, a buffer, and a culture medium. The buffer is selected from phosphate-buffered saline, HEPES, MOPS, and MES. The enzyme includes lactate dehydrogenase.

[0017] In one or more embodiments, the fluorescent probe is selected from one or more of the following kits: Bioassay Systems' EnzyFluo Lactase-Assay-Kit, Biovision's LactateAssay Kit, AAT Bioquest's Amplite Fluorescent Lactate Assay Kit, and Merck's Lactate Assay Kit. In one or more embodiments, the NAD, enzyme, buffer, and culture medium are selected from one or more of the following kits: Bioassay Systems' EnzyFluo Lactase-Assay-Kit, Biovision's Lactate Assay Kit, AAT Bioquest's Amplite Fluorescent Lactate Assay Kit, and Merck's Lactate Assay Kit.

[0018] In one or more embodiments, Bioassay Systems’ EnzyFluo Lactase-Assay-Kit is either the EnzyFluo L-Lactate Assay Kit with catalog number EFLLC-100 or the EnzyFluo D-Lactate Assay Kit with catalog number EFDLC-100.

[0019] In one or more embodiments, Biovision's Lactate Assay kit is the L-Lactate Assay Kit Colorimetric / Fluorometric (catalog number ab65330), the L-Lactate Assay Kit (Fluorometric) (catalog number ab169557), or the D-Lactate Assay Kit Fluorometric (catalog number ab174096).

[0020] In one or more embodiments, the AAT Bioquest Amplite Fluorescent Lactate Assay Kit is either the Amplite Fluorescent L-Lactate Assay Kit (catalog number 13814) or the Amplite Fluorescent D-Lactate Assay Kit (catalog number 13810).

[0021] In one or more embodiments, the fluorescent probe is the Picoprobe fluorescent probe from Biovision.

[0022] In one or more embodiments, each partition contains one or more cells. In one or more embodiments, the cells are cells that highly express lactate, such as U87 or MCF-7.

[0023] In one or more embodiments, the partition is a solution system containing a cell population.

[0024] In one or more embodiments, the partition is a partition in a droplet, a micropore array, or a porous plate. Preferably, the micropore array includes a micropore array chip, such as a micropore array chip containing micropores in a material such as glass, quartz, or PDMS; the porous plate includes 6, 24, 48, 96, 384, 1954-well plates, etc.

[0025] In one or more embodiments, step 1) includes mixing a cell phase, a lactate detection reagent phase, and an oil phase, wherein the lactate detection reagent contains a Picoprobe fluorescent probe.

[0026] In one or more embodiments, the incubation lasts for at least 5 minutes, for example, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, or at least 60 minutes.

[0027] In one or more embodiments, the cell phase is a buffer or culture medium suspension containing cells. The buffer is selected from phosphate buffer, HEPES, MOPS, and MES.

[0028] In one or more embodiments, the lactate detection reagent phase includes a Picoprobe fluorescent probe, NAD, an enzyme, a buffer solution, etc.

[0029] In one or more embodiments, the oil phase is a microdroplet-generating oil, such as fluorinated oil, mineral oil, paraffin oil, or other water-insoluble organic matter.

[0030] The present invention also provides a method for cell population, comprising:

[0031] 1) Incubate fluorescent probes and cells in separate compartments.

[0032] 2) Detect the fluorescence signal at the first emission wavelength, and distinguish between cells that secrete lactate and cells that do not contain lactate based on the difference in fluorescence signal. Cells in the region with strong fluorescence signal secrete less lactate than cells in the region with weak fluorescence signal. The first emission wavelength is 610nm-700nm.

[0033] In one or more embodiments, the cell is a leukocyte, such as a CD45-positive leukocyte.

[0034] In one or more embodiments, the fluorescent probe emits a reduced fluorescence signal at the first emission wavelength upon contact with lactic acid. Preferably, the fluorescent probe emits fluorescence at the first emission wavelength when not in contact with lactic acid, and emits essentially no fluorescence at the first emission wavelength after contact with lactic acid.

[0035] In one or more embodiments, the first emission wavelength is 620-690 nm, more preferably 630-670 nm.

[0036] In one or more embodiments, the first emission wavelength is the emission wavelength of the CY5 channel.

[0037] In one or more embodiments, the fluorescent probe emits an enhanced fluorescence signal at a second emission wavelength upon contact with lactic acid. Preferably, the fluorescent probe exhibits weak fluorescence or essentially no fluorescence at the second emission wavelength when not in contact with lactic acid, and its fluorescence is enhanced at the second emission wavelength upon contact with lactic acid, wherein the second emission wavelength is 565 nm-605 nm.

[0038] In one or more embodiments, the fluorescent probe is over-oxidized at lactic acid concentrations greater than 30 μM, preferably greater than 50 μM, greater than 80, or greater than 100 μM.

[0039] In one or more embodiments, the individual partition further comprises one, more, or all of NAD, an enzyme, a buffer, and a culture medium. The buffer is selected from phosphate-buffered saline, HEPES, MOPS, and MES. The enzyme includes lactate dehydrogenase.

[0040] In one or more embodiments, the separate partition also contains antibodies that recognize cells, such as anti-CD45 antibodies.

[0041] In one or more embodiments, the fluorescent probe is selected from one or more of the following kits: Bioassay Systems' EnzyFluo Lactase-Assay-Kit, Biovision's LactateAssay Kit, AAT Bioquest's Amplite Fluorescent Lactate Assay Kit, and Merck's Lactate Assay Kit. In one or more embodiments, the NAD, enzyme, buffer, and culture medium are selected from one or more of the following kits: Bioassay Systems' EnzyFluo Lactase-Assay-Kit, Biovision's Lactate Assay Kit, AAT Bioquest's Amplite Fluorescent Lactate Assay Kit, and Merck's Lactate Assay Kit.

[0042] In one or more embodiments, Bioassay Systems’ EnzyFluo Lactase-Assay-Kit is either the EnzyFluo L-Lactate Assay Kit with catalog number EFLLC-100 or the EnzyFluo D-Lactate Assay Kit with catalog number EFDLC-100.

[0043] In one or more embodiments, Biovision's Lactate Assay kit is the L-Lactate Assay Kit Colorimetric / Fluorometric (catalog number ab65330), the L-Lactate Assay Kit (Fluorometric) (catalog number ab169557), or the D-Lactate Assay Kit Fluorometric (catalog number ab174096).

[0044] In one or more embodiments, the AAT Bioquest Amplite Fluorescent Lactate Assay Kit is either the Amplite Fluorescent L-Lactate Assay Kit (catalog number 13814) or the Amplite Fluorescent D-Lactate Assay Kit (catalog number 13810).

[0045] In one or more embodiments, the fluorescent probe is the Picoprobe fluorescent probe from Biovision.

[0046] In one or more implementations, each partition contains one or more cells.

[0047] In one or more embodiments, the partition is a partition in a droplet, a micropore array, or a porous plate. Preferably, the micropore array includes a micropore array chip, such as a micropore array chip containing micropores in a material such as glass, quartz, or PDMS; the porous plate includes 6, 24, 48, 96, 384, 1954-well plates, etc.

[0048] In one or more embodiments, the incubation lasts for at least 5 minutes, for example, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, or at least 60 minutes.

[0049] In one or more embodiments, step 1) includes mixing a cell phase, a lactate detection reagent phase, and an oil phase, wherein the lactate detection reagent contains a Picoprobe fluorescent probe.

[0050] In one or more embodiments, the cell phase is a buffer or culture medium suspension containing cells. The buffer is selected from phosphate buffer, HEPES, MOPS, and MES.

[0051] In one or more embodiments, the lactate detection reagent phase includes a Picoprobe fluorescent probe, NAD, an enzyme, a buffer solution, etc.

[0052] In one or more embodiments, the oil phase is a microdroplet-generating oil, such as fluorinated oil, mineral oil, paraffin oil, or other water-insoluble organic matter.

[0053] The present invention also provides reagents or devices for detecting a fluorescence signal at a first emission wavelength and the use of a fluorescent probe in the preparation of a kit for detecting lactate secretion in single cells, wherein the first emission wavelength is 610 nm-700 nm.

[0054] In one or more embodiments, the fluorescence signal emitted by the fluorescent probe at the first emission wavelength is reduced upon contact with lactic acid. Preferably, the fluorescent probe emits fluorescence at the first emission wavelength when not in contact with lactic acid, and emits essentially no fluorescence at the first emission wavelength after contact with lactic acid.

[0055] In one or more embodiments, the first emission wavelength is 620-690 nm, more preferably 630-670 nm.

[0056] In one or more embodiments, the first emission wavelength is the emission wavelength of the CY5 channel.

[0057] In one or more embodiments, the fluorescent probe emits an enhanced fluorescence signal at a second emission wavelength upon contact with lactic acid. Preferably, the fluorescent probe emits essentially no fluorescence at the second emission wavelength when not in contact with lactic acid, but emits fluorescence at the second emission wavelength after contact with lactic acid, wherein the second emission wavelength is 565 nm-605 nm.

[0058] In one or more embodiments, the fluorescent probe is over-oxidized at lactic acid concentrations greater than 30 μM, preferably greater than 50 μM, greater than 80, or greater than 100 μM.

[0059] In one or more embodiments, the cells are located in separate partitions. In one or more embodiments, the partitions are partitions within a droplet, a micropore array, or a multi-well plate. Preferably, the micropore array comprises a micropore array chip, such as a micropore array chip containing micropores in a material such as glass, quartz, or PDMS; the multi-well plate comprises 6-, 24-, 48-, 96-, 384-, 1954-well plates, etc.

[0060] In one or more embodiments, each partition contains one or more cells. In one or more embodiments, the cells are cells with high lactate expression, such as U87 or MCF-7. In one or more embodiments, the cells are leukocytes.

[0061] In one or more embodiments, the fluorescent probe is a fluorescent probe selected from one or more of the following kits: Bioassay Systems' EnzyFluo Lactase-Assay-Kit, Biovision's LactateAssay Kit, AAT Bioquest's Amplite Fluorescent Lactate Detection Kit, and Merck's Lactate Detection Kit.

[0062] In one or more embodiments, Bioassay Systems’ EnzyFluo Lactase-Assay-Kit is either the EnzyFluo L-Lactate Assay Kit with catalog number EFLLC-100 or the EnzyFluo D-Lactate Assay Kit with catalog number EFDLC-100.

[0063] In one or more embodiments, Biovision's Lactate Assay kit is the L-Lactate Assay Kit Colorimetric / Fluorometric (catalog number ab65330), the L-Lactate Assay Kit (Fluorometric) (catalog number ab169557), or the D-Lactate Assay Kit Fluorometric (catalog number ab174096).

[0064] In one or more embodiments, the AAT Bioquest Amplite Fluorescent Lactate Assay Kit is either the Amplite Fluorescent L-Lactate Assay Kit (catalog number 13814) or the Amplite Fluorescent D-Lactate Assay Kit (catalog number 13810).

[0065] In one or more embodiments, the fluorescent probe is the Picoprobe fluorescent probe from Biovision.

[0066] In one or more embodiments, the reagents used to detect the fluorescence signal at the first emission wavelength include one or more selected from: NAD, enzymes, buffer solutions, culture media, microdroplet-generating oils, etc. The enzymes include lactate dehydrogenase.

[0067] In one or more embodiments, the NAD, enzyme, buffer, and culture medium are selected from one or more of the following kits: Bioassay Systems' EnzyFluo Lactase-Assay-Kit, Biovision's Lactate Assay Kit, AAT Bioquest's Amplite Fluorescence Lactate Detection Kit, and Merck's Lactate Detection Kit.

[0068] In one or more embodiments, the microdroplet generating oil includes water-insoluble organic substances such as fluorinated oil, mineral oil, and paraffin oil. Attached Figure Description

[0069] Figure 1 This is the excitation spectrum of the fluorescent probe.

[0070] Figure 2 This is the emission spectrum of the fluorescent probe.

[0071] Figure 3 To detect the signal distribution of lactic acid standards of different concentrations in droplets using the method described in this invention.

[0072] Figure 4The image shows the results of detecting high lactate-secreting cells (U87) in a droplet using the method described in this invention.

[0073] Figure 5 To use the method described in this invention, a raindrop pattern of high lactate-secreting cells (U87) was detected in a droplet.

[0074] Figure 6 The image shows the results of detecting high lactate-secreting cells (U87) in a droplet using a commercially available lactate assay kit (compared to...). Figure 4 correspond).

[0075] Figure 7 Raindrop diagram for detecting high lactate-secreting cells (U87) in a droplet using a commercially available lactate detection kit.

[0076] Figure 8 The graph shows the results of detecting high lactate-secreting cells (MCF-7) in droplets using the method described in this invention and a commercially available lactate detection kit. A1: TRITC channel 15 min; A2: TRITC channel 30 min; A3: TRITC channel 1 h; B1: CY5 channel 15 min; B2: CY5 channel 30 min; B3: CY5 channel 1 h.

[0077] Figure 9 To detect the signal distribution of high lactate-secreting cells (MCF-7) in a droplet using the method described in this invention and a commercially available lactate detection kit. A1: The method described in the lactate kit detects the lactate secreted by a single MCF-7 cell in a droplet after incubation for 15 min, 30 min, and 1 h; B1: The method described in this invention detects the lactate secreted by a single MCF-7 cell in a droplet after incubation for 15 min, 30 min, and 1 h.

[0078] Figure 10 Raindrop diagram for detecting high lactate-secreting cells (MCF-7) in droplets using the method described in this invention and a commercially available lactate detection kit. Figure 10 In the diagram, region "Ⅰ" represents an empty droplet; region "Ⅱ" represents a droplet containing MCF-7. A1: The method of this invention detects lactic acid secreted by a single MCF-7 in a droplet after 15 min of incubation; A2: The method of this invention detects lactic acid secreted by a single MCF-7 in a droplet after 30 min of incubation; A3: The method of this invention detects lactic acid secreted by a single MCF-7 in a droplet after 1 h of incubation; B1: Lactic acid reagent and the method for detecting lactic acid secreted by a single MCF-7 in a droplet after 15 min of incubation; B2: Lactic acid reagent and the method for detecting lactic acid secreted by a single MCF-7 in a droplet after 30 min of incubation; B3: Lactic acid reagent and the method for detecting lactic acid secreted by a single MCF-7 in a droplet after 1 h of incubation.

[0079] Figure 11 Fluorescence field images of CD45-labeled healthy human leukocytes encapsulated in droplets provide a reference for determining the droplet properties (empty / single-encapsulated droplets) (single-encapsulated droplets are indicated by gray circles).

[0080] Figure 12 A graph showing the results of detecting high / low lactate-secreting cells (white blood cells from healthy individuals) in a droplet using the method described in this invention (and...). Figure 11 correspond). Detailed Implementation

[0081] All references cited in this specification, including but not limited to patent publications and non-patent literature, and references cited therein, are incorporated herein by reference. The discussion of references herein is intended only to summarize the authors' assertions and does not imply that any reference constitutes prior art. The applicant reserves the right to question the accuracy and relevance of any cited references.

[0082] In this document, the term "about" or "approximately" preceding a numerical value indicates a range of that value plus or minus 10%. It should be understood that when a numerical range is given, unless otherwise expressly stated herein, all intermediate values ​​between the upper and lower limits of the range, up to one-tenth of the lower limit, and any other notations or intermediate values ​​within the range are included within the scope of this invention. The smaller ranges may independently include the upper and lower limits of these smaller ranges, which are also within the scope of this invention unless the upper and lower limits of the range are expressly excluded. When a range is defined to include one or two limits, this invention also includes ranges excluding one or both of those limits.

[0083] The phrase “and / or” as used in the specification and embodiments should be understood to mean “one or two” of the elements so joined together, that is, elements that exist together in some cases and separately in others. Multiple elements listed with “and / or” should be interpreted in the same way, that is, “one or more” elements so joined together. Other elements besides those specifically indicated by the “and / or” clause may optionally exist, whether or not they are related to those specifically indicated elements.

[0084] The inventors discovered that in commercially available single-cell lactate detection methods, if the large amount of lactate secreted by the cells causes the lactate concentration in the small-volume microchamber to be too high, almost all fluorescent probes will be over-oxidized into non-fluorescent substances. This results in no significant difference in signal between droplets of lactate-secreting cells and empty droplets without lactate, which can lead to a single high-lactate-secreting cell in the microchamber being misjudged as a low-lactate or non-lactate-secreting cell.

[0085] In attempting to solve the aforementioned problems, the inventors unexpectedly discovered that by detecting the depletion of fluorescence signals at a specific emission wavelength, the indistinguishability between droplets from lactate-secreting cells and empty droplets without lactate can be effectively avoided, significantly improving detection precision and accuracy. It has been verified that the method described in this application can achieve accurate single-cell lactate secretion detection for both standards and lactate-expressing cells.

[0086] Therefore, the present invention provides a method for detecting lactate secretion by single cells, comprising: 1) incubating a fluorescent probe and cells in separate compartments, and 2) detecting a fluorescence signal at a specific emission wavelength, wherein the fluorescence signal in the compartment containing the lactate-secreting cells is attenuated. The specific emission wavelength is 610-700 nm, preferably 620-690 nm, and more preferably 630-670 nm. The emission wavelength includes the emission wavelength of the CY5 channel. Therefore, in a specific embodiment, step 2) is: detecting the fluorescence signal of the CY5 channel.

[0087] Based on experimental results and literature data of incubating cancer cells (U87) in droplets for 60 minutes, the inventors estimated that excessive oxidation could be ensured at lactate concentrations greater than 30 mM. Therefore, the fluorescent probe was already excessively oxidized at lactate concentrations greater than 30 mM (until the positive signal was consistent with the background). Simultaneously, the maximum detectable lactate concentration of the fluorescent probe in the TRITC detection channel was 100 μM, indicating that at concentrations greater than 100 μM, excessive oxidation led to inaccurate quantitative curves in the TRITC detection channel.

[0088] In this invention, the fluorescent probe is over-oxidized at lactic acid concentrations greater than 30 μM, preferably greater than 50 μM, greater than 80 μM, or greater than 100 μM. Over-oxidation renders the fluorescence signal of the TRITC channel indistinguishable from the fluorescence signal in the absence of lactic acid. Therefore, the correlation between the TRITC channel fluorescence signal of the fluorescent probe and the lactic acid concentration weakens until it approaches the fluorescence signal in the absence of lactic acid. In this invention, the fluorescent probe emits fluorescence at an emission wavelength of 610-700 nm when not in contact with lactic acid, and the emission signal at this wavelength weakens after contact with lactic acid, and emits virtually no fluorescence at high lactic acid concentrations. Furthermore, the fluorescent probe exhibits enhanced fluorescence at a second emission wavelength after contact with lactic acid. Preferably, the fluorescent probe exhibits weak fluorescence or virtually no fluorescence at the second emission wavelength when not in contact with lactic acid, and the fluorescence enhances at the second emission wavelength after contact with lactic acid. The second emission wavelength is 565 nm-605 nm, preferably 575-595 nm. The second emission wavelength includes the emission wavelength of the TRITC channel.

[0089] In one or more embodiments, the fluorescent probe is a probe with a fluorescent signal in a lactate (fluorescence assay) kit. Exemplarily, the fluorescent probe is selected from one or more of the following kits: Bioassay Systems' EnzyFluo Lactase-Assay-Kit, Biovision's Lactate Assay Kit, AAT Bioquest's Amplite Fluorescence Assay Lactate Kit, and Merck's Lactate Assay Kit (catalog number, e.g., MAK064-1KT).

[0090] Bioassay Systems' EnzyFluo Lactase-Assay-Kit includes the EnzyFluo L-Lactate Assay Kit (product number EFLLC-100) or the EnzyFluo D-Lactate Assay Kit (product number EFDLC-100).

[0091] Biovision's Lactate Assay kits include the L-Lactate Assay Kit Colorimetric / Fluorometric (catalog number ab65330), the L-Lactate Assay Kit (Fluorometric) (catalog number ab169557), or the D-Lactate Assay Kit (Fluorometric) (catalog number ab174096). The fluorescent probes are Biovision's Picoprobe fluorescent probes.

[0092] AAT Bioquest's Amplite fluorescent lactate assay kits include the Amplite fluorescent L-lactate assay kit (catalog number 13814) or the Amplite fluorescent D-lactate assay kit (catalog number 13810).

[0093] In one or more embodiments, the separate compartment further comprises one, more, or all of NAD, enzymes, buffers, and culture media. The buffer is an isotonic buffer, such as a phosphate-based buffer or other buffer (HEPES, MOPS, MES, etc.). Exemplarily, the NAD, enzyme, and buffer are the corresponding reagents in the above-described kit. The cell culture medium is a medium suitable for culturing the cells to be tested, such as DMEM.

[0094] In some implementations, each compartment contains one cell for single-cell lactate secretion detection. A key aspect of single-cell lactate secretion detection is the need to separate individual cells into independent compartments to determine whether the lactate signal in that compartment specifically originates from that cell. In some implementations, each compartment contains multiple cells (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more). The compartments can be any single-cell detection-capable compartment known in the art, such as droplets, microwell arrays, or multiwell plates, or any compartment-generating carrier that separates cells into independent compartments. The microwell array is a microwell array chip containing micropores in materials such as glass, quartz, or PDMS, and the multiwell plate is a 6, 24, 48, 96, 384, or 1954-well plate, etc. Single cells can be separated into compartments using methods known in the art. For example, cell suspensions and detection reagents (e.g., lactate detection reagents containing fluorescent probes) can be passed through a microfluidic chip to separate single cells into microchambers.

[0095] Furthermore, the partition may also contain cell populations, such as a solution system. The method is used to detect lactate secretion signals from cell populations, thus eliminating the need to separate individual cells.

[0096] In one or more embodiments, step 1) includes incubating a mixture of a cell phase, a lactate detection reagent phase, and an oil phase, wherein the lactate detection reagent contains a Picoprobe fluorescent probe. The cell concentration of the cell phase is 1 x 10⁻⁶ cells / mL. 5 -1x10 9 / mL, preferably 1x10 6 -1x10 8 / mL, more preferably 2x10 7 -3x10 7 / mL. The incubation time is at least 5 minutes, for example, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, or at least 60 minutes. The method of the present invention can achieve high-throughput determination of high / low lactate-secreting cells in droplets by detecting the signal consumed by the fluorescent probe, which can remain stable for a relatively long time.

[0097] This invention also provides a method for cell segmentation, comprising: 1) incubating the fluorescent probe and cells described herein in separate partitions, and 2) detecting fluorescence signals at an emission wavelength of 610-700 nm, distinguishing lactate-secreting cells from non-lactic acid-secreting cells based on differences in fluorescence signals, wherein cells in partitions with strong fluorescence signals secrete less lactate than cells in partitions with weak fluorescence signals. For example, the cells may be leukocytes, such as CD45-positive leukocytes. Therefore, the separate partitions also contain antibodies that recognize the cells, such as anti-CD45 antibodies. In a specific embodiment, step 2) is: detecting the fluorescence signal of the CY5 channel.

[0098] This invention also provides reagents or devices for detecting fluorescence signals at emission wavelengths of 610-700 nm, and the use of the fluorescent probes described herein in the preparation of kits for detecting lactate secretion in single cells. Reagents for detecting fluorescence signals at said emission wavelengths include, but are not limited to, one or more of the following: NAD, enzymes, buffer solutions, cell culture media, microdroplet-generating oils, etc. Microdroplet-generating oils are known in the art, such as water-insoluble organic compounds like fluorinated oils, mineral oils, and paraffin oils.

[0099] In a specific implementation scheme, the method of the present invention is carried out through the following steps:

[0100] a. Sample collection and pretreatment: Collect cells or artificially cultured cells from samples such as plasma, pleural fluid, and urine, and add buffer solution to prepare a cell suspension of a specific concentration;

[0101] b. Preparation of lactate detection reagent: Prepare lactate detection reagent containing probe, enzyme, buffer, etc.

[0102] c. Microcompartmental separation of single cells: Cell suspension and lactate detection reagent are separated into microcompartments through a microfluidic chip;

[0103] d. Incubation: Place the microchamber containing cells in a 37°C constant temperature device and let it stand in the dark for 0 min to 6 h;

[0104] e. Signal detection: Use a microscope or other high-throughput detection equipment to acquire the signal of the corresponding channel of the fluorescent probe in the microchamber;

[0105] f. Data processing and analysis: Cells in droplets with an average fluorescence signal below a certain threshold are defined as high lactate secretion cells, and those above are defined as low lactate secretion cells.

[0106] Experiments of this invention demonstrate that by detecting the "fluorescent probe consumption" signal, the present invention can remain stable for a relatively long time (>30 min), thus enabling high-throughput determination of high / low lactate secretion cells in droplets.

[0107] Example

[0108] Unless otherwise specified, all materials and reagents used in the following examples were obtained through commercial purchase.

[0109] Reagents: Fluorescent lactate detection kit (EnzyFluo L-Lactate Assay Kit, Bioassaysystems, catalog number EFLLC-100, including fluorescent probe, buffer, and enzyme); droplet generating oil (Xinyi Manufacturing Technology Co., Ltd., microdroplet generating oil (probe method)); 1×PBS, trypsin; 5×DMEM concentrate (free of L-glutamate and sodium pyruvate); L-glutamate; sodium pyruvate; erythropoietin solution; FITC-CD45; Fc blocker.

[0110] The cells used in the experiment were human glioma cells (U87) and human breast cancer cells (MCF-7).

[0111] Example 1:

[0112] This embodiment provides a method for detecting lactic acid standards based on the probe consumption method.

[0113] I. Sample

[0114] Lactic acid standard.

[0115] II. Sample Pretreatment

[0116] Lactic acid standards were diluted to 28.6 mM and 16.6 mM using PBS.

[0117] III. Reagent Preparation

[0118] 1. Preparation of lactic acid test reagents

[0119] Prepare the commercial lactate detection working reagent according to the instructions, mixing 38 μL of 3× concentrate with 1 test lactate detection working reagent.

[0120] IV. Droplet Encapsulation

[0121] Cell phase is introduced into the reagent lactic acid standard

[0122] The reagent used in the lactate detection test is a mixture of lactate working reagent and 3×DMEM solution.

[0123] When a reagent is introduced into the oil phase, droplets are formed to generate oil.

[0124] The droplet volume is 65 pL

[0125] V. Droplet Collection and Incubation

[0126] The generated droplets were collected into 200 μL EP tubes, which were then placed in a black plastic sealed bag (to protect from light) and incubated at 37°C for 60 min.

[0127] VI. Signal Detection

[0128] After incubation, the droplets are transferred to the droplet observation chamber, and fluorescence microscopy is used to capture bright-field and CY5 channel (fluorescent probe emission channel in this detection method) signals.

[0129] VII. Test Results

[0130] Figure 3 The figure shows the average fluorescence intensity distribution of the droplet in the probe consumption method channel under different lactic acid concentrations. As can be seen from the figure, the probe signal within the droplet decreases with increasing lactic acid concentration.

[0131] Example 2:

[0132] This embodiment provides a method for detecting lactate secretion in single cells based on droplet microcavities and probe consumption.

[0133] I. Sample

[0134] U87 cells with high lactate secretion.

[0135] II. Sample Pretreatment

[0136] ①U87 cell pellet acquisition: The U87 cells adhering to the culture flask were digested with trypsin to form a U87 cell suspension, which was then transferred to a 15mL centrifuge tube and centrifuged at 1000r / min for 5min to remove the supernatant;

[0137] ② Precipitation washing: Add 2-10 mL of PBS to the precipitate, pipette tip to suspend the cells, centrifuge at 1000 r / min for 5 min to remove the supernatant;

[0138] ③ Cell lysis treatment (simulating the process of processing white blood cells in whole blood): Add 6 mL of 1× lysis solution to the precipitate, suspend it by pipetting with a disposable dropper, and lyse at room temperature for 10 min (shake gently from time to time to accelerate lysis);

[0139] ⑤ Terminate the lysis process and remove the lysis buffer (simulating the process of processing white blood cells in whole blood): After 10 min, add 6 mL of PBS to the lysis buffer to terminate the lysis. Carefully pipette the mixture evenly and centrifuge at 1200-1500 r / min for 5 min to remove the supernatant.

[0140] ⑥ Precipitation washing: Add 3-10 mL of PBS to the centrifuge tubes, resuspend the cells by pipetting, centrifuge at 1000 rpm for 5 min, and discard the supernatant. At this point, the cell count in the precipitate should be approximately 3 × 10⁻⁶ cells / mL. 6 indivual;

[0141] ⑦ Simulated antibody staining process: Transfer the cells to flow cytometry tubes, add PBS to make up to 300 μL, incubate on ice for 35-45 min, add 2 ml PBS, centrifuge at 1200-1500 rpm for 5 min, and collect the supernatant. Repeat the washing once, discard the supernatant, add PBS to the pellet to make the total volume 15 μL, so that the final cell suspension concentration is approximately 2 x 10⁻⁶. 8 cells / mL

[0142] III. Reagent Preparation

[0143] 1. Preparation of lactic acid test reagents

[0144] Prepare the commercial lactate detection working reagent according to the instructions, mixing 38 μL of 3× concentrate with 1 test lactate detection working reagent.

[0145] IV. Cell Encapsulation

[0146] The reagent introduced into the cell phase has a concentration of 2 x 10⁻⁶. 7 -3x10 7 / mL of cells in PBS suspension

[0147] The reagent used in the lactate detection test is a mixture of lactate working reagent and 3×DMEM solution.

[0148] When a reagent is introduced into the oil phase, droplets are formed to generate oil.

[0149] The droplet volume is 14 pL

[0150] V. Droplet Collection and Incubation

[0151] The generated droplets were collected into 200 μL EP tubes, which were then placed in a black plastic sealed bag (to protect from light) and incubated at 37°C for 30 min.

[0152] VI. Signal Detection

[0153] After incubation, the droplets are transferred to the droplet observation chamber, and fluorescence microscopy is used to capture bright-field and CY5 channel (fluorescent probe emission channel in this detection method) signals.

[0154] VII. Test Results

[0155] Figure 4 Images were taken after approximately 35 minutes of incubation of empty droplets and droplets containing only U87 cells. No fluorescent probe signal was observed in the droplets containing only U87 cells. Since U87 cells are highly lactate-secreting cells, the lactate secreted by the cells in the droplets containing only U87 cells consumed the fluorescent probe, resulting in a significantly lower fluorescence signal in the probe channel compared to empty droplets where the probe was not consumed.

[0156] like Figure 4 and Figure 5As shown, when using the method described in this invention for detection, the signals of droplets containing a single package of U87 and empty droplets with almost no lactic acid are clearly distinguishable, proving that this method has a good signal-to-noise ratio.

[0157] Example 3:

[0158] This embodiment provides a method for detecting lactate secretion in single cells based on droplet microchambers and commercially available reagent kits.

[0159] I. Sample

[0160] U87 cells

[0161] II. Sample Pretreatment

[0162] ①U87 cell pellet acquisition: The U87 cells adhering to the culture flask were digested with trypsin to form a U87 cell suspension, which was then transferred to a 15mL centrifuge tube and centrifuged at 1000r / min for 5min to remove the supernatant;

[0163] ② Precipitation washing: Add 2-10 mL of PBS to the precipitate, pipette tip to suspend the cells, centrifuge at 1000 r / min for 5 min to remove the supernatant;

[0164] ③ Cell lysis treatment (simulating the process of processing white blood cells in whole blood): Add 6 mL of 1× lysis solution to the precipitate, suspend it by pipetting with a disposable dropper, and lyse at room temperature for 10 min (shake gently from time to time to accelerate lysis);

[0165] ⑤ Terminate the lysis process and remove the lysis buffer (simulating the process of processing white blood cells in whole blood): After 10 min, add 6 mL of PBS to the lysis buffer to terminate the lysis. Carefully pipette the mixture evenly and centrifuge at 1200-1500 r / min for 5 min to remove the supernatant.

[0166] ⑥ Precipitation Washing: Add 3-10 mL of PBS to the centrifuge tubes, resuspend the cells by pipetting, centrifuge at 1000 rpm for 5 min, and discard the supernatant. At this point, the cell count in the precipitate should be approximately 3 x 10⁻⁶ cells / mL. 6 indivual;

[0167] ⑦ Simulated antibody staining process: Transfer the cells to flow cytometry tubes, add PBS to make up to 300 μL, incubate on ice for 40 min, add 2 ml PBS, centrifuge at 1200-1500 rpm for 5 min, and collect the supernatant. Repeat the washing once, discard the supernatant, add PBS to the pellet to make the total volume 15 μL, so that the final cell suspension concentration is approximately 2 x 10⁻⁶. 8 cells / mL

[0168] III. Reagent Preparation

[0169] 1. Preparation of lactic acid test reagents

[0170] Prepare the commercial lactate detection working reagent according to the instructions, mixing 38 μL of 3X concentrate with 1 test lactate detection working reagent.

[0171] IV. Cell Encapsulation

[0172] The reagent introduced into the cell phase has a concentration of 2 x 10⁻⁶. 7 -3x10 7 / mL of cells in PBS suspension

[0173] The reagent used in the lactate detection test is a mixture of lactate working reagent and 3X DMEM solution.

[0174] When a reagent is introduced into the oil phase, droplets are formed to generate oil.

[0175] The droplet size is 14 pL

[0176] V. Droplet Collection and Incubation

[0177] The generated droplets were collected into 200 μL EP tubes, which were then placed in a black plastic sealed bag (to protect from light) and incubated at 37°C for 30 min.

[0178] VI. Signal Detection

[0179] After incubation, the droplets were transferred to the droplet observation chamber, and bright-field and TRITC channel (recommended channel in the kit) signals were captured using a fluorescence microscope.

[0180] VII. Test Results

[0181] Figure 6 Images of empty droplets and droplets containing a single packet of U87 taken after approximately 35 minutes of incubation. The droplets in these images are similar to those in Example 2. Figure 4 One-to-one correspondence. From Figure 6 It is evident that, in commercially available reagent kits, there is no significant difference in signal between droplets containing lactate-secreting U87 cells and empty droplets without lactate; however, under the same cells and conditions, the method described in this paper still exhibits a significant signal difference.

[0182] Figure 7 Corresponding to the raindrop plot, it is also evident that the signals of empty droplets and droplets containing U87 cells completely overlap and are indistinguishable. This is because U87 cells are high-lactate-secreting cells, and the large amount of lactate they secrete results in an excessively high lactate concentration in the small microcompartments, causing almost all fluorescent probes to be over-oxidized into non-fluorescent substances. This example demonstrates that commercially available lactate kits can lead to individual high-lactate-secreting cells in microcompartments being misclassified as low / non-lactate-secreting cells.

[0183] Example 4:

[0184] This embodiment provides a comparison of evaluating the lactate secretion of single cells in microdroplet microchambers using a commercial kit method and a probe consumption method.

[0185] I. Sample

[0186] MCF-7 cells

[0187] II. Sample Pretreatment

[0188] ① Obtaining MCF-7 cell pellet: Digest the MCF-7 cells that adhere to the culture flask with trypsin to form an MCF-7 cell suspension, transfer it to a 15mL centrifuge tube, centrifuge at 1000r / min for 5min and remove the supernatant;

[0189] ② Precipitation washing: Add 2-10 mL of PBS to the precipitate, pipette tip to suspend the cells, centrifuge at 1000 r / min for 5 min to remove the supernatant;

[0190] ③ Cell lysis treatment (simulating the process of processing white blood cells in whole blood): Add 6 mL of 1× lysis solution to the precipitate, suspend it by pipetting with a disposable dropper, and lyse at room temperature for 10 min (shake gently from time to time to accelerate lysis);

[0191] ⑤ Terminate the lysis process and remove the lysis buffer (simulating the process of processing white blood cells in whole blood): After 10 min, add 6 mL of PBS to the lysis buffer to terminate the lysis. Carefully pipette the mixture evenly and centrifuge at 1200-1500 r / min for 5 min to remove the supernatant.

[0192] ⑥ Precipitation Washing: Add 3-10 mL of PBS to the centrifuge tubes, resuspend the cells by pipetting, centrifuge at 1000 rpm for 5 min, remove the supernatant, add PBS to the precipitate to make the final cell suspension concentration approximately 1 × 10⁻⁶. 7 -2×10 7 cells / mL

[0193] III. Reagent Preparation

[0194] 1. Preparation of lactic acid test reagents

[0195] Prepare the commercial lactate detection working reagent according to the instructions, mixing 38 μL of 3× concentrate with 1 test lactate detection working reagent.

[0196] IV. Cell Isolation

[0197] The cells were mixed with the lactate detection reagent, and single cells were dispersed into a microarray with a well volume of 65 μL.

[0198] V. Cell Incubation

[0199] Place the microwell array containing cells flat in a black plastic sealed bag (protected from light) and incubate at 37°C for 15 min, 30 min, and 1 h.

[0200] VI. Signal Detection

[0201] Fluorescence microscopy was used to capture bright-field, CY5 channel (fluorescent probe emission channel) signals, and TRITC (recommended channel for the kit).

[0202] VII. Test Results

[0203] Figure 8 A1, A2, and A3 are TRITC channel (recommended channel for the kit) images taken after approximately 15 min, 30 min, and 1 h of incubation for empty droplets and droplets encapsulating MCF-7, respectively. Figure 8 B1, B2, and B3 are images of the CY5 channel (the channel used in this method) taken after incubation of an empty droplet and a droplet encapsulating MCF-7 for approximately 15 min, 30 min, and 1 h, respectively. Figure 9 A1 and B1 are the average fluorescence intensity distributions of empty droplets and droplets encapsulated with MCF-7 at different incubation times (15 min, 30 min, and 1 h) corresponding to the TRITC channel (the channel recommended by the kit) and the CY5 channel (the channel used in this method). Figure 10 A1, A2, and A3 are raindrop diagrams showing the average fluorescence intensity distribution of empty droplets and droplets encapsulating MCF-7 after incubation for 15 min, 30 min, and 1 h, respectively. Figure 10 B1, B2, and B3 are raindrop diagrams showing the average fluorescence intensity distribution of empty droplets and droplets encapsulating MCF-7 after incubation for 15 min, 30 min, and 1 h, respectively.

[0204] This embodiment demonstrates that, with the commercially available lactate reagent kit method, as incubation time increases and lactate concentration in the droplets rises, the droplet signal corresponding to high lactate-secreting cells gradually weakens, leading to individual high lactate-secreting cells in the microcompartment being misidentified as low / non-lactate-secreting cells. In contrast, the method described in this patent maintains a stable droplet signal corresponding to high lactate-secreting cells at different incubation times, preventing them from being identified as low / non-lactate-secreting cells.

[0205] Example 5:

[0206] This embodiment provides a method for detecting lactate secretion in single cells based on micropores / microchambers and probe consumption.

[0207] I. Sample

[0208] U87 cells

[0209] II. Sample Pretreatment

[0210] ①U87 cell pellet acquisition: The U87 cells adhering to the culture flask were digested with trypsin to form a U87 cell suspension, which was then transferred to a 15mL centrifuge tube and centrifuged at 1000r / min for 5min to remove the supernatant;

[0211] ② Precipitation washing: Add 2-10 mL of PBS to the precipitate, pipette tip to suspend the cells, centrifuge at 1000 r / min for 5 min to remove the supernatant;

[0212] ③ Cell lysis treatment (simulating the process of processing white blood cells in whole blood): Add 6 mL of 1X lysis solution to the precipitate, suspend it by pipetting with a disposable dropper, and lyse at room temperature for 10 min (shake gently from time to time to accelerate lysis);

[0213] ⑤ Terminate the lysis process and remove the lysis buffer (simulating the process of processing white blood cells in whole blood): After 10 min, add 6 mL of PBS to the lysis buffer to terminate the lysis, carefully pipette the mixture evenly, centrifuge at 1200-1500 r / min for 5 min and remove the supernatant;

[0214] ⑥ Precipitation washing: Add 3-10 mL of PBS to the centrifuge tubes, resuspend the cells by pipetting, centrifuge at 1000 rpm for 5 min, and discard the supernatant.

[0215] ⑦ Simulated antibody staining process: Transfer the cells to flow cytometry tubes, add PBS to make up to 300 μL, incubate on ice for 1 hour, add 2 ml of PBS, centrifuge at 1200-1500 rpm for 5 minutes, and collect the supernatant. Repeat the washing once, discard the supernatant, and add PBS to the pellet to make the final cell suspension concentration approximately 10. 6 -10 7 cells / mL

[0216] III. Reagent Preparation

[0217] 1. Preparation of lactic acid test reagents

[0218] Prepare the commercial lactate detection working reagent according to the instructions, mixing 38 μL of 3× concentrate with 1 test lactate detection working reagent.

[0219] IV. Cell Isolation

[0220] Mix the cells with the lactate detection reagent, disperse single cells into a microarray with a well volume of 1 nL. V. Cell Incubation

[0221] Place the microwell array containing cells flat in a black plastic bag (protected from light) and incubate at 37°C for 6 hours.

[0222] VI. Signal Detection

[0223] Fluorescence microscopy captures bright-field and CY5 channel (fluorescent probe emission channel) signals.

[0224] Example 6:

[0225] This embodiment provides a method for further distinguishing cell subpopulations using the method described in this invention in conjunction with antibody labeling.

[0226] I. Sample

[0227] Fresh whole blood sample from healthy individuals

[0228] II. Sample Pretreatment

[0229] ① Sample collection: Whole blood from healthy individuals is collected into anticoagulant tubes and transported at 4°C. The time difference between blood sample collection and processing should not exceed 30 minutes.

[0230] ② Sample pretreatment: Take 500 μL of whole blood from healthy individuals into a 15 mL centrifuge tube, add 2 mL of PBS, mix well with pipette tip, and centrifuge to remove the supernatant (orange-yellow plasma layer);

[0231] ③ Cell lysis treatment: Add 4 mL of 1× lysis solution to the precipitate, resuspend by pipetting with a single dropper, and lyse at room temperature for 10 min (shake gently from time to time to accelerate lysis);

[0232] ⑤ Terminate the lysis process and remove the lysis buffer: After 10 min, add 6 mL of PBS to the lysis buffer to terminate the lysis. Carefully pipette the mixture evenly and centrifuge at 1200-1500 r / min for 5 min to remove the supernatant.

[0233] ⑥ Precipitation washing: Add 3-10 mL of PBS to the above mixture, pipette to suspend the cells, centrifuge at 1000 rpm for 5 min to remove the supernatant. At this point, the cell count in the precipitate should be approximately 3 × 10⁻⁶ cells. 6 indivual;

[0234] ⑦ Fc antibody blocking: The above cells were transferred to flow cytometry tubes, and PBS was added to make up to 300 μL. 15 μL of Fc antibody was added to the tubes and the cells were placed on ice for 10 min.

[0235] ⑧ Antibody staining: Add 60 μL of FITC-CD45 antibody to the Fc antibody-blocked system and incubate on ice for 25 min. After incubation, add 2 mL of PBS to the system, centrifuge at 1200-1500 rpm for 5 min, and collect the supernatant. Repeat the washing once, discard the supernatant, and add PBS to the pellet to a final volume of 15 μL, making the cell suspension concentration 2 × 10⁻⁶. 7 cells / mL

[0236] III. Reagent Preparation

[0237] 1. Preparation of lactic acid test reagents

[0238] Prepare the commercial lactate detection working reagent according to the instructions, mixing 38 μL of 3× concentrate with 1 test lactate detection working reagent.

[0239] IV. Cell Encapsulation:

[0240] The reagent introduced into the cell phase has a concentration of 2 x 10⁻⁶. 7 -3x10 7 / mL of cells in PBS suspension

[0241] The reagent used in the lactate detection test is a mixture of lactate working reagent and 3×DMEM solution.

[0242] When a reagent is introduced into the oil phase, droplets are formed to generate oil.

[0243] V. Droplet Collection and Incubation

[0244] The generated droplets were collected into 200 μL EP tubes, which were then placed in a black plastic sealed bag (to protect from light) and incubated at 37°C for 10 min.

[0245] VI. Signal Detection

[0246] After incubation, the droplets are transferred to the droplet observation chamber, and fluorescence microscopes are used to capture bright-field, CY5 channel (emission channel of the fluorescent probe), and FITC channel (corresponding to the CD45 signal) signals.

[0247] VII. Test Results

[0248] Since almost all leukocytes express CD45, CD45 conjugated with fluorescent molecules can be used to label leukocytes. In this example, FITC-conjugated CD45 is used to label leukocytes, which are then encapsulated in droplets. The droplets with the CD45 signal are shown below. Figure 11 As shown in the figure, the droplets with fluorescent spots (marked with gray circles) are droplets containing CD45 positive cells.

[0249] Furthermore, after incubating FITC-CD45-labeled leukocytes in droplets for 1 hour, the lactate secretion was detected using the method described in this invention, as shown below. Figure 12 As shown, it is similar to Figure 11 The displayed droplets correspond one-to-one. Figure 11 Droplets of CD45-positive cells, some of which were in Figure 12 The fluorescence signal of the Cy5 channel in the droplet was significantly lower than that in the empty droplet, indicating that the droplet contained highly lactate-secreting cells expressing CD45.

[0250] As shown in the figure, in CD45-positive cells, this method can be used to further distinguish between low-lactate-secreting cells with no decreased fluorescence signal and high-lactate-secreting cells with significantly decreased fluorescence signal. Therefore, the method of this invention can accurately detect cellular lactate secretion levels and differentiate cell subpopulations.

[0251] Example 7:

[0252] This embodiment provides a fluorescence emission wavelength range that can detect probe signals.

[0253] I. Sample

[0254] probe, PBS

[0255] II. Reagent Preparation

[0256] PBS dilution of probes forms probe dilution solution

[0257] III. Signal Detection

[0258] The probe dilution solution was added to a quartz dish, and the excitation and emission spectra of the probe were measured using a fluorescence spectrophotometer.

[0259] IV. Test Results

[0260] Figure 1 , Figure 2 These are the excitation and emission spectra of the probe, respectively.

[0261] The probe exhibits the strongest fluorescence signal at its optimal emission wavelength. The further the probe deviates from the optimal emission wavelength, the weaker the signal becomes, until it becomes indistinguishable from the background. Therefore, referencing the standards used in analytical chemistry to distinguish a signal from the background signal (three times the background fluctuation coefficient of variation plus the average background value is the minimum value that can be distinguished from the background), we estimated the range of emission wavelengths in which the probe can be distinguished from the background.

[0262] We added three times the background fluctuation coefficient of the background value detected in the CY5 channel to obtain the lowest fluorescence value that can be distinguished from the background. After converting it into the relative fluorescence intensity in the fluorescence emission spectrum, we substituted this value into the emission spectrum to estimate the range of emission wavelengths higher than this value.

[0263] The lowest measurable relative fluorescence intensity can be calculated as follows: L = (M1 + 3SD) × M4 ÷ [M × (M2 / M3)].

[0264] In the formula, M is the average fluorescence intensity of the droplet encapsulating the probe under a fluorescence microscope; M1 is the fluorescence intensity of the oil (background) generated by the droplet under a fluorescence microscope; SD is the corresponding standard deviation; M2 is the fluorescence intensity value corresponding to the optimal excitation wavelength of the probe under a fluorescence spectrophotometer; M3 is the fluorescence intensity value at the corresponding wavelength of the microscope filter under a fluorescence spectrophotometer; and M4 is the fluorescence intensity value at the optimal emission wavelength of the probe under a fluorescence spectrophotometer.

[0265] We calculated that L = 19. Substituting the emission spectrum data, we found that when the relative fluorescence intensity near the optimal emission wavelength is 19, the corresponding emission wavelengths are 620 nm and 690 nm, respectively. Therefore, we estimate that the detectable probe emission wavelength range is 610 nm-700 nm, and at least 620 nm-690 nm.

Claims

1. A method of detecting cell lactate secretion, comprising: 1) incubating a fluorescent probe and cells in separate partitions, 2) detecting a fluorescent signal at a first emission wavelength, wherein the fluorescent signal is attenuated in the partitions where lactate is secreted by the cells, the first emission wavelength being between 610 nm and 700 nm, wherein the fluorescent probe emits fluorescence at the first emission wavelength when not in contact with lactate, and the fluorescent signal is attenuated at the first emission wavelength when in contact with lactate, and the fluorescent probe emits fluorescence at a second emission wavelength when in contact with lactate, the fluorescent probe being weakly fluorescent or not fluorescent at the second emission wavelength when not in contact with lactate, and the fluorescent signal is enhanced at the second emission wavelength when in contact with lactate, the second emission wavelength being between 565 nm and 605 nm, and the fluorescent probe is over-oxidized at a lactate concentration greater than 30 μΜ; and the fluorescent probe is selected from one or more of the following kits: EnzyFluo Lactase-Assay-Kit from Bioassay system, Lactate Assay from Biovision, Amplite Fluorimetric Lactate Assay Kit from AAT Bioquest, Lactate Assay Kit from Merck.

2. The method of claim 1, wherein the fluorescent probe is over-oxidized at a lactate concentration greater than 50 μΜ.

3. The method of claim 1 or 2, wherein, the fluorescent probe is Picoprobe fluorescent probe from Biovision.

4. The method of claim 1 or 2, wherein, the separate partitions further comprise one, more or all of NAD, enzyme, buffer, media.

5. The method of claim 4, wherein, the buffer is selected from phosphate buffer, HEPES, MOPS, MES, and / or the enzyme is lactate dehydrogenase.

6. The method of claim 4, wherein, the NAD, enzyme, buffer, media are selected from one or more of the following kits: EnzyFluo Lactase-Assay-Kit from Bioassay system, Lactate Assay from Biovision, Amplite Fluorimetric Lactate Assay Kit from AAT Bioquest, Lactate Assay Kit from Merck.

7. The method of claim 1 or 2, wherein, the partitions are droplets, microwell arrays, or partitions in a multi-well plate.

8. The method of claim 1 or 2, wherein, Step 1) comprises mixing a cell phase, a lactate detection reagent phase, and an oil phase, wherein the lactate detection reagent comprises the fluorescent probe.

9. The method of claim 8, wherein the cell phase is a suspension of cells in a buffer or media, the buffer being phosphate buffer, HEPES, MOPS, MES.

10. The method of claim 8, wherein the lactate detection reagent phase comprises the fluorescent probe, NAD, enzyme, and buffer, and / or the oil phase is a microdroplet generating oil, comprising a fluorinated oil, a mineral oil, or a paraffin oil.

11. A method of cell sub-population, comprising: 1) incubating a fluorescent probe and cells in separate partitions, 2) detecting a fluorescent signal at a first emission wavelength, wherein the fluorescent signal is stronger in the subregion with lower secretion of lactic acid than in the subregion with higher secretion of lactic acid, and wherein the first emission wavelength is between 610 nm and 700 nm, wherein the fluorescent probe emits fluorescence at the first emission wavelength when not in contact with lactic acid, and wherein the fluorescent signal at the first emission wavelength is reduced when in contact with lactic acid, and wherein the fluorescent probe emits weak fluorescence or substantially no fluorescence at a second emission wavelength when not in contact with lactic acid, and wherein the fluorescent signal at the second emission wavelength is increased when in contact with lactic acid, and wherein the second emission wavelength is between 565 nm and 605 nm, and wherein the fluorescent probe is over-oxidized at a lactic acid concentration greater than 30 μΜ, and wherein the fluorescent probe is selected from one or more of the following kits: EnzyFluo Lactase-Assay-Kit by Bioassay system, Lactate Assay Kit by Biovision, Amplite Fluorimetric Lactic Acid Assay Kit by AAT Bioquest, Lactate Assay Kit by Merck.

12. The method of claim 11, wherein the fluorescent probe is over-oxidized at a lactic acid concentration greater than 50 μΜ.

13. The method of claim 11, wherein, the fluorescent probe is Picoprobe fluorescent probe by Biovision.

14. The method according to any one of claims 11 to 13, wherein, the individual subregion further comprises one, more, or all of NAD, enzyme, buffer, medium.

15. The method of any one of claims 11-13, wherein, the individual subregion further comprises an antibody that recognizes the cell.

16. The method of claim 14, wherein, the buffer is selected from phosphate buffer, HEPES, MOPS, MES, and / or the enzyme is lactate dehydrogenase.

17. The method of claim 14, wherein the NAD, enzyme, buffer, medium are selected from one or more of the following kits: EnzyFluo Lactase-Assay-Kit by Bioassay system, Lactate Assay Kit by Biovision, Amplite Fluorimetric Lactic Acid Assay Kit by AAT Bioquest, Lactate Assay Kit by Merck.

18. The method of any one of claims 11-13, wherein, the subregion is a droplet, a microwell array, or a well of a multi-well plate.

19. The method of any one of claims 11-13, wherein, Step 1) comprises mixing a cell phase, a lactic acid detection reagent phase, and an oil phase, wherein the lactic acid detection reagent comprises the fluorescent probe.

20. The method of claim 19, wherein the cell phase is a suspension of cells in a buffer or medium, and wherein the buffer is phosphate buffer, HEPES, MOPS, MES.

21. The method of claim 19, wherein the lactic acid detection reagent phase comprises the fluorescent probe, NAD, enzyme, and buffer, and / or the oil phase is a microdroplet-generating oil comprising a fluorinated oil, a mineral oil, or a paraffin oil.

22. Use of a reagent or device for detecting a fluorescent signal at a first emission wavelength, the first emission wavelength being 610 nm - 700 nm, and a fluorescent probe in the manufacture of a kit for detecting lactic acid secretion by a single cell, wherein the fluorescent probe emits fluorescence at the first emission wavelength in the absence of lactic acid, the fluorescent signal at the first emission wavelength is reduced after contact with lactic acid, and the fluorescent probe emits a fluorescent signal at a second emission wavelength after contact with lactic acid, the second emission wavelength being 565 nm - 605 nm, and the fluorescent probe is over-oxidized at lactic acid concentrations greater than 30 μΜ.

23. The use of claim 22, wherein the fluorescent probe is over-oxidized at lactic acid concentrations greater than 50 μΜ.

24. The use of claim 22, wherein the fluorescent probe is a Picoprobe fluorescent probe from Biovision.

25. The use of claim 22, wherein the reagent for detecting a fluorescent signal at a first emission wavelength comprises one or more of the following selected from the group consisting of: NAD, an enzyme, a buffer, a medium, an oil phase.

26. The use of claim 25, wherein the oil phase is a microdroplet generating oil comprising a fluorinated oil, a mineral oil, or a paraffin oil. ​ ​ ​ ​ ​ 25. Use according to any one of claims 22 to 24, characterized in that, ​ 26. The use of claim 25, wherein, ​

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