Method for measuring oxidative stress

Abstract

A method of measuring oxidative stress in a cell or tissue sample, the method comprising the steps of: (i) treating the cell or tissue sample in a culture medium; (ii) collecting a portion of the culture medium; (iii) measuring the amount of reduced glutathione (GSH) and oxidized glutathione (GSSG) in the cell or tissue sample; and (iv) measuring the amount of reactive oxygen species (ROS) in the culture medium portion collected in step (ii); wherein step (iii) is performed immediately after steps (i) and (ii); and wherein step (iv) is performed concurrently with step (iii), or within 6 hours after step (iii) is completed if the collected medium portion is stored at about 4 DEG C prior to performing step (iv), including the determination time.

Description

TECHNICAL FIELD

[0001] The present invention relates to a method of measuring oxidative stress in cells or tissues. In particular, a method is described in which the amount of reduced and oxidized glutathione and the amount of reactive oxygen species are measured in the same cell or tissue sample at the same time point. BACKGROUND

[0002] Oxidative stress is a common marker of environmental factor toxicity and is also fundamental to intrinsic cellular processes. Oxidative stress can be described as a redox imbalance in biological systems due to an excessive rate of production of oxidizing species, in particular reactive oxygen species (ROS), which cannot be counteracted by the antioxidant defenses, including reduced glutathione (GSH), which when oxidized becomes glutathione disulfide (GSSG). This difference can cause damage to cells and cellular systems (Vona R, et al. The Impact of Oxidative Stress in Human Pathology: Focus on Gastrointestinal Disorders. Antioxidants (Basel). 2021 Jan 30;10(2):201. doi: 10.3390 / antiox10020201. PMID: 33573222; PMCID: PMC7910878) and oxidative stress has been shown to play an important role in the pathogenesis and progression of many human diseases (Forman HJ, Zhang H. Targeting oxidative stress in disease: promise and limitations of antioxidant therapy. Nat Rev Drug Discov. 2021 Sep;20(9):689-709. doi: 10.1038 / s41573-021-00233-1. Epub 2021 Jun 30. Erratum in: Nat Rev Drug Discov. 2021 Aug;20(8):652. PMID: 34194012; PMCID: PMC8243062).

[0003] Most studies on oxidative stress provide an incomplete characterization of this cellular process according to its scientifically accepted definition, as only ROS or glutathione (GSH and / or GSSG) amounts are evaluated, and not both biomarkers in the same biological sample at the same time point. The relevance of the available data on oxidative stress in the literature is often controversial due to lack of precision resulting from the use of different biological samples, different time points, and low response time resolution (e.g. Sies H, et al., Defining roles of specific reactive oxygen species (ROS) in cell biology and physiology. Nat Rev Mol Cell Biol. 2022 Jul;23(7):499-515 PMID: 35190722, and for a summary, Frijhoff J, et al., Clinical Relevance of Biomarkers of Oxidative Stress. Antioxid Redox Signal. 2015 Nov 10;23(14):1144-40. PMID: 26415143).

[0004] Due to low throughput capacity and sensitivity to interference of the tested compounds, most available methods to measure oxidative stress are not compatible with 3D tissue models, nor with high throughput evaluation activities such as screening.

[0005] The present invention seeks to provide an improved method to measure oxidative stress, wherein both ROS and glutathione (GSH and GSSG) amounts can be accurately measured in the same tissue at the same time point. SUMMARY

[0006] The present inventors have developed a method to measure oxidative stress in a cell or tissue sample by optimizing bioluminescence-based tests. Unexpectedly, they found that two different tests can be implemented simultaneously in the same sample. That is, two tests are performed at the same endpoint for a comprehensive and biologically relevant characterization of oxidative stress.

[0007] First, the present inventors have utilized a luciferin-NT substrate and a glutathione S-transferase (e.g. GSH-Glo®) to measure the amount of ROS and GSH in the same sample at the same time point. TMThe assays (determinations) were improved. Specifically, they improved such assays by including an additional step that was not part of the known protocol and used a reducing agent capable of reducing disulfide bonds (e.g., dithiothreitol (DTT), dithioerythritol, dithionite, sodium borohydride and potassium borohydride, tris(2-carboxyethyl)phosphine (TCEP), triphenylphosphine and tributylphosphine as described in, e.g., Monostori P, Wittmann G, Karg E, Turi S. Determination of glutathione and glutathione disulfide in biological samples: an in-depth review. J Chromatogr B Anal Technol Biomed Life Sci 2009; 877:3331-46. 10.1016 / j.jchromb.2009.06.016) to not only quantify GSH (reduced form) but also total glutathione and GSSG (oxidized form) in the same sample. This optimization allowed calculation of the GSH / GSSG ratio, which is an informative measure of cellular redox state and related oxidative stress biomarkers. Although alternative kits exist for GSH / GSSG ratio assessment (e.g., Promega GSH / GSSG-Glo TM ), the improved method described herein provides a more relevant ratio measurement by assessing both GSH (reduced form) and GSSG (oxidized form) from the same biological sample, and has the added benefit of reducing costs by fifty percent in terms of sample requirements.

[0008] Second, the inventors also supplemented the GSH / GSSG assay with assessment of another oxidative stress biomarker, ROS. This can be used on the same sample as the glutathione assay and thus enables a more holistic understanding of the oxidative stress of a cell or tissue sample at a particular time point. As used herein, the term "biomarker" refers to a molecule, gene, or characteristic that can identify a particular physiological process, especially a pathological process.

[0009] The present invention allows measurement of multiple oxidative stress biomarkers over a certain time course (e.g., on a cell or tissue sample or on an individual from which cells are harvested at various time points over a time period in which oxidative stress occurs). Time course is important in this context due to the dynamic and transient nature of biological phenomena (Lushchak Ukr. Biochem. J. 2015; Vol. 87, No. 6, November-December, pp. 11-18).

[0010] In a first aspect, a method of measuring oxidative stress in a cell or tissue sample is described, the method comprising the steps of: (i) treating the cell or tissue sample in a culture medium; (ii) collecting a portion of the culture medium; (iii) measuring the amount of glutathione (GSH, GSSG and total glutathione) in the cell or tissue sample; and (iv) measuring the amount of reactive oxygen species (ROS) in the portion of culture medium collected in step (ii); wherein step (iii) is performed immediately after steps (i) and (ii); and wherein step (iv) is performed simultaneously with step (iii), or within 6 hours (including assay time) after step (iii) is completed if the collected portion of culture medium is stored at between 0 and 8°C, preferably between 2 and 6°C, most preferably at about 4°C prior to performing step (iv).

[0011] Suitably, the amount of ROS and / or the amount of glutathione is measured using luminescence.

[0012] Suitably, the amount of ROS and / or the amount of glutathione is measured quantitatively or qualitatively or relatively.

[0013] Suitably, the culture medium of step (i) of the method comprises a hydrogen peroxide (H202) substrate, most suitably a ROS-Glo® H202 substrate. TM H202 substrate.

[0014] Suitably, the treatment in step (i) of the method lasts for 6 hours or less.

[0015] Suitably, the amount of glutathione measured in step (iii) of the method is the sum of GSH (reduced form) and GSSG (oxidized form).

[0016] Suitably, the amount of glutathione measured in step (iii) takes into account the stoichiometry of the glutathione redox cycle, wherein 2 GSH (reduced) molecules are oxidized to a single GSSG (oxidized) molecule, and vice versa, a single GSSG molecule is reduced back to two GSH molecules.

[0017] Suitably, a GSH / GSSG ratio is calculated in step (iii) of the method.

[0018] Suitably, the GSH / GSSG ratio is calculated using relative luminescence units (RLU) or glutathione molarity from a glutathione standard curve.

[0019] Suitably, a reducing agent (suitably a reducing agent capable of reducing disulfide bonds) is used to measure the total glutathione amount in step (iii) of the method.

[0020] In a further aspect, step (iii) of the method comprises at least the steps of: -

[0021] (a) placing the cell or tissue sample in a well plate, adding GSH-Glo TM reagent to each well and shaking at about 50-350 rpm, preferably about 300 rpm, for about 2-10 minutes, preferably about 5 minutes, at room temperature to lyse the cells;

[0022] (b) adding GSH-Glo TM reagent to standard curve wells in the assay plate;

[0023] (c) adding GSH-Glo TM reagent to reductant wells in the assay plate and to one or more water vehicle background control wells;

[0024] (d) dividing each well containing lysed cell or tissue sample into two wells in the assay plate;

[0025] (e) to measure total GSH, adding reductant to one of each pair of sample wells and to at least one reductant background control well;

[0026] (f) to measure reduced GSH, adding water to the remaining sample wells without reductant and to the one or more water vehicle background control wells;

[0027] (g) shaking all wells at about 300 rpm for about 1 minute at room temperature;

[0028] (h) incubating all wells at room temperature;

[0029] (i) adding reagent capable of revealing signal from the luciferin-luciferase system to sample wells, to at least one reductant well and one or more water vehicle background control wells, and to standard curve wells;

[0030] (j) shaking all wells at about 300 rpm for about 1 minute at room temperature;

[0031] (k) incubating all wells at room temperature; and

[0032] (l) measuring luminescence.

[0033] Suitably, the reductant is capable of reducing disulfide bonds, suitably the reductant is dithiothreitol (DTT), dithioerythritol, dithionite, sodium borohydride, potassium borohydride, tris(2-carboxyethyl)phosphine (TCEP), triphenylphosphine, or tributylphosphine, for example TCEP Bond breaker TM tris(2-carboxyethyl)phosphine (TCEP).

[0034] Suitably, step (1) is performed using a luminescence plate reader.

[0035] In yet another aspect, step (iii) of the method comprises at least the following steps:

[0036] (A) removing cell supernatant from the sample;

[0037] (B) adding a passive lysis buffer to the sample and shaking at about 50-350 rpm, preferably about 300 rpm, for about 2-10 minutes, preferably 5 minutes;

[0038] (C) dividing the sample equally into two wells;

[0039] (D) adding luciferin-NT to one well to quantify total glutathione (GSH + GSSG);

[0040] (E) adding luciferin-NT and N-ethylmaleimide (NEM) to the other well to quantify oxidized GSSG;

[0041] (F) incubating both wells at room temperature with shaking at about 300 rpm for about 5 minutes;

[0042] (G) adding luciferin generation reagent to each well. Shake for about 1 minute;

[0043] (H) incubating both wells at room temperature;

[0044] (I) adding reagents to both wells that reveal the signal from the luciferin-luciferase system and shaking for about 1 minute;

[0045] (J) incubating both wells for about 14 minutes;

[0046] (K) transferring the sample wells to an assay plate containing a calibration curve; and

[0047] (L) measuring luminescence.

[0048] Suitably, step (L) is performed using a luminescence plate reader

[0049] Optionally, the buffer in step (B) is kept cold. For example, 0-6°C. Optionally, the buffer in step (B) is kept on ice. Optionally, the buffer used in step (B) is double concentrated (2X) to facilitate lysis of tissue samples.

[0050] In yet another aspect, step (iv) of the method described herein comprises at least the following steps:

[0051] (a) adding a media only control sample and an equivalent amount of a compound interference control to an assay plate;

[0052] (b) adding a standard curve to at least 10 wells of the assay plate;

[0053] (c) adding to all wells a reagent capable of revealing the signal from the luciferin-luciferase system, if D-cysteine is not yet present in the reagent, adding D-cysteine, preferably 0.01 to 20 mM, preferably 0.1 to 10 mM;

[0054] (d) incubating the assay plate for about 1 minute at room temperature with shaking at about 300 rpm;

[0055] (e) incubating the assay plate for about 20 minutes at room temperature.

[0056] (f) measuring luminescence.

[0057] Suitably, wherein step (f) is performed using a luminescence plate reader, suitably using a pre-set "ROS-Glo TM H2O2 Assay".

[0058] An alternative signal enhancer solution can optionally be added on top in step (c) to enhance the signal, such as Pierce TM Firefly Signal Enhancer (Thermofisher) (https: / / www.thermofisher.com / order / catalog / product / 16180).

[0059] Still another aspect of the application comprises a kit comprising materials for carrying out one or more methods of the application, and optionally instructions. Suitably, the materials comprise reagents required for one or more methods of the application.

[0060] In still another aspect, the kit of the application comprises a GSH-Glo TM Assay from catalog # V6911 or V6912 as described in Technical Manual # TB369 from Promega, in addition or alternatively, the kit comprises a GSH-Glo TM Assay from catalog V6912 as described in Technical Manual # TB344 from Promega, plus a reducing agent, and optionally instructions. Suitably, the reducing agent is capable of reducing disulfide bonds, suitably the reducing agent is dithiothreitol (DTT), dithioerythritol, dithionite, sodium borohydride, potassium borohydride, tris(2-carboxyethyl)phosphine (TCEP), triphenylphosphine, or tributylphosphine, suitably the reducing agent is TCEP.

[0061] Suitably, the kit of the application further comprises a ROS-Glo TMMaterials for H2O2 determination and optional further instructions. Suitably, the kit includes materials from catalogs #G8820 and G8821 of the TM391 ROS-Glo kit as described in Promega's technical manual TM391. TM H2O2 assay kit.

[0062] Some advantages

[0063] This invention has the advantage of enabling a comprehensive assessment of oxidative stress in tissue or cell samples by evaluating the levels of two major oxidative stress biomarkers, ROS and the GSH / GSSG ratio, in the same sample at the same time point.

[0064] This invention allows for multiple measurements of the oxidative stress biomarkers within a defined timeframe or period. For example, measurements can be performed on cell or tissue samples at multiple time points within a period of oxidative stress, or on individuals from whom cell samples were collected. For multiple time points, ROS assays require only one sample if a non-lytic variant is used, but glutathione assays require multiple samples (one for each time point).

[0065] This method can be applied to 2D and 3D cell culture models for time-series experiments and can be used in a high-throughput manner.

[0066] Compared to other methods, the methods of the present invention are less susceptible to compound interference in the context of chemical evaluation because they are based on luminescence readings rather than most available fluorescence or colorimetric readings. Attached Figure Description

[0067] Figure 1 A figure comparing known methods for assessing oxidative stress with the method of the present invention.

[0068] Figure 2 This diagram illustrates the conversion of luciferin derivatives to luciferin in the presence of glutathione, a conversion catalyzed by glutathione S-transferase.

[0069] Figure 3 The known GSH-Glo is illustrated in diagram form. TM Comparison of the determination (top, part A) with the method of the present invention (bottom, part B).

[0070] Figure 4A To demonstrate the use of GSH-Glo TM A graph showing the amount of GSH (reduced form) measured in the sample by the kit.

[0071] Figure 4B To illustrate the use of the modified GSH-Glo in the method of the present invention TMGraph of the amounts of GSH, GSSG and combined GSH + GSSG measured in the sample by the kit.

[0072] Figure 4C Graph showing the GSH / GSSG ratio obtained from the results shown in Figure 4B .

[0073] Figure 5 Graph showing how the GSH / GSSG and ROS assays were adapted to be compatible with the 3D bronchial cultures. TM Non-lysis protocol.

[0074] Figure 6 Graph showing the results obtained from the experimental protocol. TM H202assay.

[0075] Figure 7 Graph showing the experimental protocol to obtain the results shown in Figure 8 .

[0076] Figure 8 The top row shows the results of the improved GSH / GSSG-Glo TM kit and ROS-Glo TM H202assay on the same sample at different time points. In the top row, we see a comparison of the standard (non-improved) assay on the same cell culture at the same time point.

[0077] Figure 9 Graph showing the dose-dependent effect on glutathione levels, ROS and cell viability when the improved GSH / GSSG-Glo Figure 8 kit and ROS-Glo TM H202assay were performed on the same samples as shown in TM .

[0078] Figure 10A Graph showing the characteristics of fully differentiated 3D bronchial cultures from primary human cells, including cell type composition and in vitro culture conditions. This was adapted from Kirkpatrick and Millard, BioRxiv . 2020 https: / / doi.org / 10.1101 / 2020.09.16.300483.

[0079] Figure 10B Close-up of one of the sections of the Figure 10A .

[0080] Figure 11 Another schematic showing how the GSH / GSSG and ROS assays were adapted to be compatible with the 3D bronchial cultures.

[0081] Figure 12 A set of graphs showing experimental data obtained by using the method of the application on 3-D bronchial cultures.

[0082] Figure 13 A set of graphs showing experimental data obtained by using the method of the application on A549 cell line.

[0083] Figure 14 A schematic representation of the experimental protocol to show the results obtained Figure 15

[0084] Figure 15 Further experimental data obtained by using the method of the application on 3d bronchial cultures are shown. DETAILED DESCRIPTION

[0085] ​The practice of the present disclosure employs, unless otherwise indicated, conventional techniques of engineering, microengineering, microbiology, cell biology, and biochemistry. Such techniques are explained fully in, for example, Molecular Cloning: A Laboratory Manual, Second Edition (Sambrook et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (MJ. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J. E. Cellis, ed., 1998) Academic Press; Animal Cell Culture (R.I. Freshney, ed., 1987); Introduction to Cell and Tissue Culture (J. P. Mather and P.E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, IB. Griffiths, and D.G. Newell, eds., 1993-8) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Current Protocols in Molecular Biology (F.M. Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994). Procedures employing commercially available kits and reagents will generally be used according to the manufacturer’s determined protocols, unless otherwise indicated.

[0086] Technical terms and expressions used herein are generally intended as applied to their meaning in the relevant field of molecular biology, microbiology, cell biology, and biochemistry. All the following term definitions apply throughout the present application.

[0087] As used herein, the singular forms “a,” “an,” and “the” include both singular and plural referents unless the context clearly dictates otherwise.

[0088] The term “and / or” means (a) or (b) or both (a) and (b).

[0089] As used herein, the term "comprising" is synonymous with "including," "containing," and "comprehending," and is inclusive or open-ended and does not exclude additional, unrecited members, elements, or method steps.

[0090] The term "consisting of means excluding other components and only having the recited elements and no other elements.

[0091] Numerical range recitations in this application include all values and subranges between the recited limits.

[0092] As used herein, when referring to measurable values such as parameters, amounts, time periods, and the like, the term "about" means that variations of + / - 10% or less, preferably + / - 5% or less, more preferably + / - 1% or less, and still more preferably + / - 0.1% or less, of the specified value are encompassed by the term, so long as such variations are appropriate in the context of the disclosure. It is to be understood that the value to which the modifier "about" refers is itself explicitly and preferably disclosed.

[0093] While the term "one or more", such as one or more members of a group of members, is explicit by itself, by way of further exemplification, the term encompasses inter alia a reference to any one of the recited members, or to any two or more of the recited members, e.g., to any >3, >4, >5, >6, or >7, etc. of the recited members, and up to all of the recited members.

[0094] The term "at least" includes no less than the stated minimum, but can include more / additional steps or items.

[0095] A first aspect of the present application is a method of measuring oxidative stress in a cell or tissue sample, the method comprising the steps of: (i) treating the cell or tissue sample in a culture medium; (ii) collecting a portion of the culture medium; (iii) measuring the amount of glutathione in the cell or tissue sample; and (iv) measuring the amount of reactive oxygen species (ROS) in the portion of culture medium collected in step (ii); wherein step (iii) is performed immediately after steps (i) and (ii); and wherein step (iv) is performed simultaneously with step (iii), or within 6 hours (including assay time) of completion of step (iii) if the collected portion of culture medium is stored at about 1-6°C, suitably about 4°C, prior to performing step (iv). The portion of culture medium collected in step (ii) contains culture medium and cells.

[0096] ​As used herein, the term "oxidative stress" refers to a disturbance in the balance between the production of reactive oxygen species (herein referred to as "ROS") and the antioxidant defenses in a cell that detoxify the reactive species or repair damage caused by them. In other words, this occurs when the normal redox state of the cell is disturbed, and this can cause toxic effects through the production of free radicals that can damage cellular components such as DNA and disrupt cellular functions such as respiration (see, e.g., Lushchak Ukr. Biochem. J. 2015; Vol. 87, Issue 6, Nov-Dec, pp. 11-18). Oxidative stress can also lead to lipid peroxidation, protein carbonylation, and apoptosis (cell death). A "free radical" is any molecular species capable of independent existence that contains an unpaired electron in an atomic orbital. ROS are a subset of free radicals that contain oxygen. Examples of ROS include superoxide radical (O2 - ), hydroxyl radical (OH), and hydrogen peroxide (H2O2).

[0097] Oxidative stress can be caused by environmental hazards such as overeating, but it is also part of many cellular processes such as oxidative phosphorylation. Oxidative stress is believed to be associated with the development of many diseases in humans, especially neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease, and multiple sclerosis. However, ROS can have beneficial uses in the immune system. There are many methods of measuring oxidative stress, some of which are outlined in Table 1 below.

[0098] Table 1

[0099] As referred to herein, "GSH" is the reduced form of glutathione made from three amino acids: cysteine, glutamate, and glycine, which has the structure shown below.

[0100] All animal cells are capable of producing GSH, but in mammals, GSH is primarily produced by the liver. GSH is involved in many bodily processes, such as tissue building and repair, making chemicals and proteins needed by the body, and immune system function.

[0101] As referred to herein, "glutathione disulfide" or "GSSG" is the oxidized state of GSH, which is a disulfide derived from two glutathione molecules, which has the following structure.

[0102] (image by Fvasconcellos 23:40, 1 October 2007 (UTC) - Own work, Public Domain, https: / / commons.wikimedia.org / w / index.php?curid=2843592)

[0103] In living cells, glutathione disulfide is reduced to two glutathione molecules, with the reducing equivalent coming from coenzyme NADPH.

[0104] GSH protects cells by reducing ROS. This transformation is illustrated by the reduction of peroxide, as shown in the following equation: 2 GSH + R2O2→ GSSG + 2 ROH (R = H, alkyl)

[0105] This reaction can be catalyzed by enzymes such as glutathione peroxidase and peroxiredoxin.

[0106] The term "glutathione" can refer to either GSH (reduced form) or GSSG (oxidized form), but unless otherwise specified refers herein to the sum of reduced GSH and oxidized GSSG.

[0107] The GSH:GSSG ratio is an important biomarker of cell health, with a decreased ratio indicating oxidative stress.

[0108] The terms "cell culture" and "tissue culture" are used interchangeably herein. Cell culture generally refers to the removal of cells from a tissue (i.e., a cell or tissue sample) and then growing in an artificial environment. The cells to be cultured can be removed directly from a tissue containing the cells to be cultured, and optionally treated with enzymes or mechanically prior to culture. As an alternative, the cells to be cultured can be derived from a previously established strain or cell line. Sections from cells or tissues from cultures to be tested are referred to herein as "cell samples" or "tissue samples" or "biological samples," and these terms are used interchangeably herein. A "cell line" is a collection of cells derived from one cell that can be propagated indefinitely in culture.

[0109] The present disclosure utilizes a variety of cell sources. In one embodiment, the present disclosure excludes a step of isolating or obtaining a cell sample from a subject. Cells can be cryopreserved. Cells can be in a 3-dimensional cell culture. Cells can be in the form of a tissue. Cells can be in the form of a spheroid. Cells can be actively dividing. Cells can be cultured in a cell culture device in the presence of a cell culture medium (e.g., comprising nutrients (e.g., proteins, peptides, amino acids), energy (e.g., carbohydrates), essential metals and minerals (e.g., calcium, magnesium, iron, phosphate, sulfate), buffers (e.g., phosphate, acetate), pH change indicators (e.g., phenol red, bromocresol purple), selective agents (e.g., chemicals, antimicrobials), etc.). A single cell culture medium can be used to grow cells of the same or different types. Different cell culture media can be used to grow cells of different types. Mixing of different cell culture media will occur as the cell culture media is circulated according to the present disclosure. In some experiments herein, “NHBE cells” were used. “NHBE” stands for “normal human bronchial epithelial” cells. These are primary cells isolated from the epithelial lining of the airways above the lung bifurcation. The NHBE cells are commonly used in respiratory disease research. In some experiments herein, an “A549” cell line (also referred to as “A549 cells”) was used. This is a cell line of human adenocarcinoma alveolar basal epithelial cells of lung tissue origin. This cell line is commonly used in the development of drug therapies, especially cancer therapies.

[0110] In some embodiments, one or more reagents are included in a cell culture medium or a plurality of cell culture media. Cells can be isolated from a tissue or fluid using methods well known in the art. Cells can be differentiated from stem cells (such as embryonic stem cells or induced pluripotent stem cells) or directly from somatic cells. Cells can be native cells or variant cells (e.g., cells comprising one or more non-native genetic variations). Cells can be disease cells or disease model cells. For example, cells can be cancer cells or cells that can be induced into a hyperproliferative state (e.g., transformed cells). The terms “medium,” “media,” and “culture medium / media” are used interchangeably herein. In some steps of the methods described herein, the culture medium used comprises a hydrogen peroxide (H2O2) substrate. As used herein, the term “substrate” refers to an addition to a culture medium that can act as a probe, e.g., by inducing a color change or luminescence. Most suitably, the culture medium used herein comprises a substrate that reacts directly with H2O2 to produce luminescence, most suitably a ROS-Glo TM H2O2substrate. The ROS-Glo TM H2O2substrate reacts directly with H2O2 to produce a luciferin precursor.

[0111] The cells can be or can be derived from a human or animal subject, or from human or animal cells, including any of a number of mammalian species, suitably human, but including rat, mouse, pig, rabbit, and non-human primate, among others. Cells and cell lines can be obtained from commercial sources. The cells can be from or derived from any desired tissue or organ type, including but not limited to adrenal gland, bladder, blood vessels, bone, bone marrow, brain, cartilage, cervix, cornea, endometrium, esophagus, gastrointestinal system, immune system (e.g., T lymphocytes, B lymphocytes, white blood cells, macrophages, and dendritic cells), liver, lung, lymphatic system, muscle (e.g., cardiac muscle), nervous system, ovary, pancreas (e.g., pancreatic islet cells), pituitary gland, prostate, kidney, salivary gland, skin, tendon, testes, and thyroid.

[0112] Lung cells, including lung epithelial cells, are a particularly interesting cell type. Bronchial and / or other airway epithelial cells are especially useful in the present disclosure. Human bronchial epithelial cells can be collected by brushing the donor lung during a bronchoscopy procedure. In one embodiment, the lung cells are Normal Human Bronchial Epithelial (NHBE) cells. Lung epithelial cells can be cultured as a monolayer of undifferentiated cells, or further developed into organotypic lung epithelial cell-like tissue at an air-liquid interface. Lung epithelial cells can be obtained from human or animal subjects having different pathologies, including subjects classified as smokers or non-smokers.

[0113] In one embodiment, the cells used are hepatocytes. Hepatocytes are the cells of the liver, which constitute 70-85% of the liver cytoplasmic mass. The functionality of hepatocytes is highly dependent on their ability to form a polarized phenotype, which is only established in 3-dimensional culture. One source of hepatocytes is primary hepatocytes, which are an in vitro model widely used to study many aspects of liver physiology and pathology. Techniques for isolating human hepatocytes can be based on a two-step collagenase perfusion of a donated liver. However, these cells express metabolic enzymes for no more than 5 days. Another limitation is their short viability. These drawbacks can be overcome by using alternative, long-lived hepatocyte lines, such as human or animal hepatoprogenitor cell lines. One such example of a human hepatoprogenitor cell line is HepaRG TM HepaRG cells (ThermoFisher Scientific). HepaRG TM The cells retain many characteristics of primary human hepatocytes. They have higher liver-specific and metabolic gene expression, and have a longer lifespan, compared to primary hepatocytes. HepaRG TMRecombinant cells further extend lifespan and improve metabolic capacity, suggesting that spheroids can provide a better surrogate in vitro liver model for toxicity testing. Liver spheroids can also be created with mixtures of primary hepatocytes with hepatic stellate cells or primary hepatocytes with adipose tissue-derived stem cells.

[0114] In one embodiment, the lung cell is a lung epithelial cell, such as a bronchial and / or other airway epithelial cell.

[0115] In one embodiment, the liver cell is a hepatocyte, suitably a HepaRG cell.

[0116] The use of combinations of any of the cells described herein is envisaged. Any combination of cells described herein is used in the methods of the application. One exemplary combination of cells is a combination of liver cells and lung cells. The use of lung epithelial cells, such as bronchial and / or other airway epithelial cells, in combination with liver cells, such as HepaRG cells, is envisaged. TM Combinations of cells. Other cells can be used with the combination, if desired.

[0117] The different cells of the combination can be cultured in separate wells.

[0118] The present disclosure incorporates the use of "3-dimensional cell culture" (or "3D cell culture" used interchangeably herein), which includes any method that provides for the culture of cells in 3-dimensions, with or without the use of a matrix or scaffold, such as a permeable membrane in an insert. A number of different 3-dimensional cell culture methods have been developed, including spheroid culture and organotypic culture. 3-dimensional cells can be grown and / or maintained in the cell culture devices described herein. The tissue or cell samples used in any of the methods described herein can be cultured in 3-dimensional cell culture prior to, during or after the methods of the application are performed.

[0119] The term "spheroid" takes its meaning as generally understood in the art, which is a single cell that divides into a cell sphere in 3-dimensions, or an aggregate of multiple cells in 3-dimensions, supported with or without a matrix or scaffold in 3-dimensional cell growth within the spheroid. The three-dimensional spheroid can be an adherent spheroid or a spheroid grown in suspension.

[0120] In some embodiments, the spheroid comprises a single cell type. In some embodiments, the spheroid comprises more than one cell type. In some embodiments, when more than one spheroid is grown, each spheroid is of the same type, while in other embodiments, two or more different types of spheroids are grown.

[0121] 3-dimensional spheroids more closely resemble in vivo tissue in terms of their cell communication and development of extracellular matrix.In vivo The matrix helps cells move within the spheroids, similar to how cells move in living tissue. Therefore, spheroids are a highly improved model for differentiation, survival, cell migration, cell polarization, gene expression, and growth.

[0122] Spherical bodies can be harvested and studied using a variety of methods well-known in the field, including colorimetric, fluorescence, and luminescence analyses measured with plate readers, or those readily observable by microscopy. Other techniques include Western, Northern, or Southern blotting, histological techniques (e.g., immunohistochemistry, in situ hybridization, immunofluorescence), etc. The use of optical imaging methods, such as reverse bright-field microscopy and fluorescence microscopy, is also envisioned.

[0123] Applications of 3D spheres include in vitro cell and tissue proliferation studies in environments closer to the in vivo environment, compound screening, toxicology testing, cell therapy, cell delivery, reagent delivery, biochemical substitution, generation of bioactive molecules, tissue engineering, biomaterials, and clinical trials.

[0124] The use of spheroids in three-dimensional cell culture is generally in Expert Opin. Drug Discov A review was conducted in (2015)10, 519–540.

[0125] 3D organ culture systems (especially those in miniature form) can be used in this disclosure because they allow for the study of how organs function at a microscale. Responses to certain stimuli, responses to one or more reagents, and the pharmacokinetic behavior of such reagents can be studied. Miniature 3D cell culture systems allow for the combined study of cell groups or organ groups. This allows for the reproduction of the complexity of interactions between different tissues. 3D organ cultures can be organoid, meaning they attempt to reproduce the main functions of an organ or organ system. Miniature fluid systems that interconnect pores are also envisioned.

[0126] On one hand, a spheroid culture is provided, wherein the spheroids exist as individual spheroids after 5 hours of culture or after 5 days of culture. In other words, the spheroids do not aggregate or fuse. The culture can be used as a cell or tissue sample in any of the methods described herein.

[0127] Liver The liver plays a vital role in detoxification, the metabolism of carbohydrates, lipids, and proteins, and the biotransformation of endogenous and exogenous substances. Liver function is closely linked to the assembly of highly specialized cells (mostly hepatocytes) embedded within a complex three-dimensional structure known as the "lung lobes." Biotransformation of compounds typically produces non-toxic and more soluble metabolites; however, occasionally, more toxic metabolites are formed, leading to hepatotoxicity.

[0128] Hepatocytes can be kept 3-dimensional via various methods, including the use of sandwich culture, solid scaffold materials such as polystyrene scaffolds, hydrogels such as type I collagen, or self-assembly into spheroids.

[0129] While the use of freshly isolated primary human hepatocytes can be the preferred hepatocyte type, their availability is limited. Other options for human hepatocyte lines include HepG2 and Hep2 / C3A cells. One particularly suitable cell source is HepaRG TM Cell lines. Other sources of human hepatocytes are human embryonic stem cell (hESC)-derived hepatocytes and hepatocytes derived from induced pluripotent stem cells (iPSCs).

[0130] In one embodiment, the spheroids are or are derived from hepatocytes to form 3- dimensional liver spheroids. Such liver spheroids can be made using methods known in the art and described, for example, in ALTEX (2014) 31, 441-477 and Toxicol. Sci . (2013) 133, 67-78.

[0131] Lung — As the morphology of the respiratory tract changes from the upper to the lower airways, many different cell culture models have been established using primary airway epithelial cells or cell lines and encompassed for use in the present disclosure. The choice of which cells or cell lines to use exactly will depend on the region of the respiratory tract of interest for the given study.

[0132] As the lung surface is exposed to air, cell models can be cultured at the air-liquid interface to more realistically mimic the lung.

[0133] In one embodiment, the lung 3-dimensional cultures are or are derived from lung cells to form 3-dimensional organotypic tissues. Such lung tissues can be made using various methods known in the art, such as described in ALTEX (2014) 31, 441-477 and Toxicol . (2013) 133, 67-78.

[0134] The cell cultures used in the disclosed methods can also be cultured on plates in regular 2-dimensional cell culture. For example, in a Petri dish.

[0135] As used herein, the term "treatment" refers to the addition of any chemical, nutrient, or composition to a cell sample or culture as an alternative or in addition to changing any environmental condition such as temperature. Treatments can be added, for example, via bath or pour. Treatments can be added to a culture medium, or the treatment can comprise a specific culture medium. For example, in some embodiments, the methods described herein treat the tissue with a culture medium that directly reacts with H202 to generate a luciferin precursor

[0136] Treatments can be basolateral (applied to the base, basal membrane, or outside of a cell or tissue) or apical (applied to the top, outside, or air-facing surface of a cell or tissue. Basolateral treatments can allow for better control of chemical compound concentrations. However, in some embodiments, apical treatments are more physiologically relevant, for example, for air-liquid interface 3D tissues or submerged tissues.

[0137] As used herein, the term "collect" refers to removing all or some of a sample, liquid, or solid. For example, removing some cells from a culture for use in the next step of a method.

[0138] As used herein, the term "portion" refers to any part of a whole. 1%, 2%, 5%, 10%, 20%, 25%, 30%, 50%, 75%, 80%, or any amount can constitute a portion.

[0139] As used herein, the term "measure" refers to any way of determining the size, amount, or degree of something. Measurements (to produce a measurement value) can be quantitative or qualitative or relative. Measurement values can be quantitative, qualitative, or relative.

[0140] In particular, in the methods described herein, it refers to measuring the amount of ROS and / or the amount of glutathione (GSH and GSSG or both). These measurements are indicative of oxidative stress as discussed above.

[0141] In some embodiments of the methods described herein, the amount of ROS and / or the amount of glutathione is measured quantitatively or qualitatively or relatively. In some embodiments, the amount of ROS and / or the amount of GSH is measured using luminescence. The methods can measure GSH (reduced), GSSG (oxidized), or total glutathione.

[0142] The measurements described herein, suitably luminescence measurements, can be relative to a standard curve or background,

[0143] In some embodiments, the luminescence is measured quantitatively or qualitatively or relatively.

[0144] As used herein, the term "amount" refers to the quantity of something, especially the sum in number, size, value, or degree of one or more things.

[0145] As used herein, the expression "immediately after" means consecutively within a short time, suitably within 10 minutes, suitably within 30 minutes, suitably within 60 minutes.

[0146] As used herein, the term "simultaneously" means at the same time or within 5, 10, 20, 30 minutes or less.

[0147] Suitably, the treatment in step (i) of the method described herein lasts for 6 hours or less, preferably 5 hours or less, preferably 4 hours or less, preferably 3 hours or less, preferably 2 hours or less, most preferably 1 hour or less.

[0148] As used herein, the term "luminescence" refers to the emission of light by a substance due to a chemical or enzymatic reaction (bioluminescence). Luminescence detection is optically simpler than fluorescence detection, as it does not require a light source or specific optical elements to excite.

[0149] The glutathione assays of the present application are luminescence-based assays for the detection and quantification of GSH and / or GSSG. They are based on the conversion of a luciferin derivative to luciferin in the presence of glutathione, which is catalyzed by glutathione S-transferase (GST). The signal generated in a coupled reaction with firefly luciferase is proportional to the amount of glutathione present in the sample. Figure 2 The reactions involved are illustrated.

[0150] Suitably, the amount of glutathione measured in step (iii) of the method is the sum of reduced GSH and oxidized GSH (GSSG).

[0151] Suitably, the GSH / GSSG ratio is calculated in step (iii) of the method.

[0152] Suitably, the GSH / GSSG ratio is calculated using relative luminescence units (RLU). RLU is the unit used for most luminescence measurements. RLU has no physical meaning and is generally not comparable between different instruments.

[0153] Suitably, a reducing agent is used to measure the GSH amount in step (iii) of the method. Suitably, the reducing agent is capable of reducing disulfide bonds, suitably the reducing agent is dithiothreitol (DTT), dithioerythritol, dithionite, sodium borohydride, potassium borohydride, tris(2-carboxyethyl)phosphine (TCEP), triphenylphosphine or tributylphosphine. Suitably, the reducing agent is TCEP, suitably Bond breaker TM TCEP.

[0154] As used herein, the term "room temperature" is about 18-25 °C, suitably about 18-22 °C, suitably about 20-22 °C.

[0155] As used herein, the term "incubate" refers to a period of time in which a sample and / or reagents are allowed to progress and / or react and / or develop. Incubation can be performed in the dark or in otherwise attenuated light.

[0156] In yet another aspect, step (iii) of the method of the present application comprises at least the following steps:

[0157] (a) placing the cell or tissue sample in a well plate, adding GSH-Glo TM reagent to each well and shaking at about 300 rpm for about 5 minutes at room temperature to lyse the cells;

[0158] (b) adding GSH-Glo TM reagent to standard curve wells in the assay plate;

[0159] (c) adding GSH-Glo TM reagent to reductant wells in the assay plate and to one or more water vehicle background control wells;

[0160] (d) dividing each well containing lysed cell or tissue sample into two wells in the assay plate;

[0161] (e) adding reductant to one of each pair of sample wells and to at least one reductant background control well for measurement of total glutathione;

[0162] (f) adding water to the remaining sample wells without reductant and to the one or more water vehicle background control wells for measurement of reduced GSH;

[0163] (g) shaking all wells at about 300 rpm for about 1 minute at room temperature;

[0164] (h) incubating all wells at room temperature;

[0165] (i) adding reagent capable of revealing signal from the luciferin-luciferase system to sample wells, to at least one reductant and one or more water vehicle background control wells, and to standard curve wells;

[0166] (j) shaking all wells at about 300 rpm for about 1 minute at room temperature;

[0167] (k) incubating all wells at room temperature; and

[0168] (l) measuring luminescence.

[0169] Suitably, the shaking in step (a) can be for about 250-350 rpm, most suitably 390-310 rpm, for about 1 to about 8 minutes.

[0170] Suitably, the shaking in step (j) can be for about 50-350 rpm, most suitably 390-310 rpm, for about 10 seconds to about 5 minutes, preferably 1-2 minutes.

[0171] Suitably, a reducing agent is used in steps (c), (e), (f) and (i) of the method. Suitably, the reducing agent is capable of reducing disulphide bonds, suitably the reducing agent is dithiothreitol (DTT), dithioerythritol, dithionite, sodium borohydride, potassium borohydride, tris(2-carboxyethyl)phosphine (TCEP), triphenylphosphine or tributylphosphine. Suitably, the reducing agent is TCEP, suitably Bond breaker TM TCEP.

[0172] Suitably, step (l) is performed using a luminescent plate reader, suitably using a pre-set protocol.

[0173] Suitably, the "GSH-Glo TM Reagent" is glutathione S transferase (for use in the GST reaction buffer in the Promega GSH-Glo TM assay and its kit (cat # V6911, V6912, V6611 and V6612). Alternatively, this can be replaced with recombinant GST and its reaction buffer from any other supplier. It is necessary to add Luciferen-NT substrate to this reaction buffer. The alternative to Luciferen-NT present in the same Promega kit can be produced by a strategy of caging the luciferin with a nitrophenylsulfonamide group.

[0174] As used herein, "Luciferase Detection Reagent" refers to Promega product cat # V8921 or V8920. It can be replaced with equivalent reagents from other suppliers, including luciferase, esterase, ATP and Mg2+, which were developed to reveal the signal from the luciferin-luciferase (Ultra-Glow Luciferase) system.

[0175] Luciferase Detection Reagent can also be replaced with firefly luciferase (0.0001 to 10 units / mL, preferably 0.1 to 1 unit / mL) diluted in a buffer solution such as 1 x Tris-Glycine buffer (pH 7.6) or 0.1 M Tris-HCl (pH 8), with the following components added:

[0176] • EDTA 0.1 mM - 2 mM

[0177] • Mg in the form of MgS04 2+ (8 - 16 mM)

[0178] • ATP: 0.1 to 10 mM, preferably 1 to 5 mM

[0179] • Esterase: 10 -6 to 10 -2 units / mL, preferably 10 -4 to 10 -3 units / mL

[0180] The individual components can be obtained separately or already in the form of a kit.

[0181] In a further aspect, step (iii) of the method comprises at least the following steps:

[0182] (A) removing the cell supernatant from the sample;

[0183] (B) adding a passive lysis buffer to the sample and shaking at about 50-350 rpm, preferably 300 rpm, for about 2-10 minutes, preferably about 5 minutes;

[0184] (C) dividing the sample equally into two wells;

[0185] (D) adding luciferin-NT to one well to quantify total GSH;

[0186] (E) adding luciferin-NT and N-ethylmaleimide (NEM) to the other well to quantify oxidized GSSG;

[0187] (F) incubating both wells at room temperature with shaking at about 300 rpm for about 5 minutes;

[0188] (G) adding luciferin generation reagent to each well. Shake for about 1 minute;

[0189] (H) incubate both wells at room temperature;

[0190] (I) add reagents to both wells that reveal the signal from the luciferin-luciferase system and shake for about 1 minute;

[0191] (J) incubate both wells for about 14 minutes;

[0192] (K) transfer the sample wells to an assay plate containing a calibration curve; and

[0193] (L) measure luminescence.

[0194] Suitably, the shaking in steps (B) and (F) can be at about 50-350 rpm, most suitably 300-310 rpm, for about 2 to about 10 minutes.

[0195] Suitably, method steps B to E are performed under low temperature conditions, such as on ice, suitably at about 1-10 °C, suitably at about 1-5 °C, most suitably at about 4 °C.

[0196] Suitably, the shaking in steps G and I is performed at 50-350 rpm, most suitably 300 rpm.

[0197] Suitably, step (L) is performed using a luminescent plate reader.

[0198] As used herein, "passive lysis buffer" is a specially formulated to facilitate rapid lysis of cultured cells, suitably mammalian cells, without the need to scrape adherent cells or perform freeze-thaw cycles. For example, Passive Lysis Buffer 5X from Promega, Cat #E1941, elicits only a minimal amount of coelenterazine autofluorescence, making it particularly suitable for assays such as those described herein.

[0199] As used herein, "luciferin generation reagent" is a buffer that can be prepared with purified glutathione-S-transferase, 2.5 mM dithiothreitol (DTT) (available from many suppliers) without using other available routes by which it is obtained from Promega kits. It is diluted in Dulbecco's Phosphate Buffered Saline or 100 mM NaH2P04(pH 7.0) and 1 mM EDTA.

[0200] As used herein, "calibration curve" is a plot of how an instrument responds to an analytical signal. It is a method of determining the concentration of a substance in an unknown sample by comparing it to a set of standard samples of known concentrations. It can also be referred to as a standard curve. In the assays disclosed herein, multiple wells in any sample plate can be assayed in order to determine a calibration curve with which to compare the results (typically luminescence) of a cell or tissue sample. For example, for the methods described herein, a standard curve is prepared using GSH standard solutions (glutathione, 5 mM) to facilitate the conversion of luminescence (units RLU) to GSH and / or GSSG concentration. A 5 mM glutathione stock (1 : 100) is diluted in water and then serial 1 : 1 dilutions are performed by combining equal volumes of glutathione and equal volumes of water. 10 μL of each dilution standard is transferred to the appropriate well for assay. The final concentration of 5 mM glutathione will range from 0 μM to 5 μM.

[0201] The general principles of calibration curve preparation are set forth in:

[0202] FDA Bioanalytical Method Validation Industry Guidance

[0203] https: / / www.fda.gov / files / drugs / published / Bioanalytical-Method-Validation-Guidance-for-Industry.pdf

[0204] and / or

[0205] ICH Guideline M10 on the Validation of Bioanalytical Methods and Analysis of Research Samples - https: / / www.ema.europa.eu / en / documents / scientific-guideline / ich-guideline-m10-bioanalytical-method-validation-step-5_en.pdf

[0206] Chemicals that can be used for calibration curve preparation include glutathione (e.g., Merck catalog number Y0000517, Thermo Scientific). TM Catalog number 78259, Spectrum catalog number GL146, or the kind available in the Promega kits discussed herein) and H2O2 for ROS calibration curves (e.g., Sigma H1009, ThermoScientific).

[0207] In another aspect, step (iv) of the method described herein includes at least the following steps: -

[0208] (a) Add a control sample containing only the culture medium and an equal amount of the compound interference control to the assay plate;

[0209] (b) Add the standard curve to at least 10 wells of the test plate;

[0210] (c) Add a reagent to all wells that can reveal signals from the luciferin-luciferase system;

[0211] (d) Shake the measuring plate at approximately 300 rpm for about 1 minute at room temperature;

[0212] (e) Incubate the test plate at room temperature for about 20 minutes.

[0213] (f) Measure the luminescence.

[0214] Appropriately, step (f) is performed using a light-emitting plate reader.

[0215] Suitablely, the oscillation in step (d) can be about 250-350 rpm, most preferably 300-310 rpm, for about 10 seconds to about 5 minutes, or suitablely about 30 seconds to about 2 minutes.

[0216] Another aspect of the invention includes a kit comprising GSH-Glo from Promega. TM The analyte, along with a reducing agent capable of reducing disulfide bonds such as dithiothreitol (DTT), dithioerythritol, dithionite, sodium borohydride, potassium borohydride, tris(2-carboxyethyl)phosphine (TCEP), triphenylphosphine or tributylphosphine, and the instruction manual.

[0217] Suitablely, the kit of the present invention also contains ROS-Glo from Promega. TM H2O2 analytes.

[0218] The methods described herein were tested using menadione to induce oxidative stress in cells. Menadione (vitamin K3) is a synthetic analog of 1,4-naphthoquinone with a methyl group at the 2-position. Natural menadione (vitamin K3) is lipid-soluble, while sodium menadione bisulfite is the synthetic form of menadione and is water-soluble. Menadione is used as a phosphatase inhibitor and a mitochondrial DNA polymerase γ (pol γ) inhibitor. Menadione can also be used as an inducer of oxidative damage.

[0219] The method described in this article is to combine and modify two commercially available kits from Promega for assays.

[0220] GSH / GSSG determination is based on GSH / GSSG-Glo TM The assay kits are cataloged as #V6611 and V6612 and described in the technical manual from Promega #TM344. This manual is available here.

[0221] https: / / web.archive.org / web / 20230421070742 / https: / ch.promega.com / - / media / files / resources / protocols / technical-manuals / 101 / gsh-gssg-glo-assay-protocol.pdf?rev=cfd04feb992845f298e63d80cc9c4ed8&sc_lang=en

[0222] An alternative to this method can be used with GSH-Glo TM The glutathione assay kits catalog #V6911 and V6912 operate and are described in the technical manual from Promega #TB369. This manual is available here.

[0223] https: / / ch.promega.com / - / media / files / resources / protocols / technical-bulletins / 101 / gsh-glo-glutathione-assay-protocol.pdf?rev=2aa5c77ea6a840b39aeecd94ebb5473b&sc_lang=en

[0224] As mentioned above, a reducing agent is added to these methods and the sample is split in two to enable the calculation of the amount of GSH and GGSH from the same sample.

[0225] The ROS endpoint assays described herein are based on the ROS-Glo® TM The non-lytic variant of the H202assay kit catalog #G8820 and G8821 and described in Technical Manual TM391 from Promega. This manual is available here.

[0226] https: / / ch.promega.com / - / media / files / resources / protocols / technical-manuals / 101 / ros-glo-h2o2-assay-protocol.pdf?rev=f40c6e59ae684980b36441078f53a480&sc_lang=en

[0227] Where reference is made herein to "assays from Promega", it is intended to refer to one and / or the kit comprising the necessary reagents and / or instrumentation and / or instructions of these assays.

[0228] The combination of the ROS non-lytic (non-destructive) variant of the protocol allows to run the assay on the same sample with the GSH / GSSH assay. It also allows a multi-time course characterization of the ROS by collecting a sample of the culture medium at each time point to be evaluated. These multi-time course sampling methods have not been described previously.

[0229] The application is also described in the following examples, which are provided to describe the preferred modes of practicing the application in more detail. These examples set forth presently preferred modes of practicing the application, which are intended to be illustrative and not limiting of the application.

[0230] Example

[0231] Example 1

[0232] Tissue exposure and endpoint sample collection

[0233] 1. Subject the tissue sample to basolateral treatment in culture medium containing 25 µM ROS-Glo TM H2O2substrate for the desired exposure duration.

[0234] Due to substrate instability, treatment duration is no more than 6 hours.

[0235] 2. After treatment, collect 75 µL of medium for the ROS endpoint (see Example 3) and transfer the remaining cell or tissue sample to a new 24-well plate for the GSH / GSSG endpoint. Take into account the treatment duration, the ROS samples are stored at 4°C for a total of 6 hours, while the GSH / GSSG samples must be processed immediately.

[0236] Example 2

[0237] GSH / GSSG Assay

[0238] This particular example is based on the GSH-Glo TM Assay Kit Catalog # V6911 and V6912 and described in Technical Bulletin # TB369 from Promega.

[0239] 1. Starting with the cell or tissue sample from point 2 of Example 1, add 100 µL GSH-Glo TM (1x) reagent to each tissue insert in a 24-well plate and shake at 300 rpm for 5 minutes at room temperature.

[0240] 2. Add 100 µL GSH-Glo TM (1x) reagent to the standard curve wells in the assay plate.

[0241] 3. Add 25 µL GSH-Glo TM Tris(2-carboxyethyl)phosphine (TCEP) (Thermo Fisher Scientific, Waltham, MA) to the Bond breaker TM (1x) reagent to the one or more water vehicle background control wells.

[0242] 4. Divide the tissue lysate (100 µL) from each well into two wells (25 µL each) in the assay plate, one for the addition of TCEP and the other for the addition of water.

[0243] 5. Add 2.5 µL Bond breaker TM TCEP to one of the lysate well pairs to measure total GSH and the same TCEP to the TCEP background control well.

[0244] 6. Add 2.5 μL H20 to the lysate well of the other of the lysate well pair to measure reduced GSH, and the same H20 to the one or more water vehicle background control wells.

[0245] 7. Shake the plate at 300 rpm for 1 minute at room temperature.

[0246] 8. Incubate the plate at room temperature for 24 minutes.

[0247] 9. Add 25 μL luciferin detection reagent to all sample wells and to the TCEP and water vehicle background control wells and 100 μL luciferin detection reagent to the standard curve wells.

[0248] 10. Shake the plate at 300 rpm for 1 minute at room temperature.

[0249] 11. Incubate the plate at room temperature for 14 minutes.

[0250] 12. Measure luminescence, preferably using the pre-set "GSHG Assay" protocol with an Omega.

[0251] Example 3

[0252] GSH / GSSG endpoint analysis

[0253] 1. Calculate relative luminescence units (RLU) by subtracting the appropriate background RLU from the measured raw RLU.

[0254] 2. Calculate GSSG RLU using the following formula: GSSG = (total glutathione RLU - reduced GSH RLU) / 2., where total GSH is taken from the TCEP treated sample and reduced GSH is taken from the water treated sample. During the reduction reaction, 1 mole of GSSG produces 2 moles of GSH equivalent. Therefore, dividing the GSH signal by two gives the signal for GSSG.

[0255] 3. Use the calculated GSSG RLU and measured GSH RLU to determine the GSH / GSSG ratio.

[0256] Results for Examples 1-3 are shown in Figure 4B and 4C . Figure 4A Results using the unmodified GSH-Glo TM assay and kit are shown for comparison.

[0257] Oxidative stress is induced by adding menadione to the cell or tissue sample prior to performing the assay.

[0258] Reference GSH / GSSG ratios for different cell types by Promega are provided in Table 1 below.

[0259] Table 1

[0260] Figure 4A 、 4B and 4C show significant variability in quantifying GSH levels with the GSH-Glo TM kit. Unexpectedly, low GSH and high GSSG levels were detected with the modified (TCEP added) GSH-Glo TM kit. The resulting GSH / GSSG ratio was very low for vehicle. The overall variability and low GSH compared to GSSG and total glutathione can be due to auto-oxidation of GSH during sample processing.

[0261] Similar results were obtained with 3D bronchial epithelial cells (data not shown).

[0262] Example 4

[0263] GSH / GSSG Assay - Alternative Method

[0264] This particular example is based on the GSH / GSSG-Glo TM Assay Kit Catalog # V6611 and V6612 and described in Technical Manual # TM344 from Promega

[0265] (A) Start with cell or tissue sample from point 2 from Example 1, remove cell supernatant.

[0266] (B) Add 100 μL of cold passive lysis buffer diluted 1x in water to each well of the tissue / cell sample and shake at 300 rpm for 5 minutes on ice.

[0267] (C) Place 100 μL of lysate into 2 wells, 50 μL per well.

[0268] (D) Add 1 μL of luciferin-NT to one well to quantify total GSH.

[0269] (E) Add 1 μL of luciferin-NT and 0.5 μL of 25 mM N-ethylmaleimide (NEM) to the other well to quantify oxidized GSSG.

[0270] (F) Incubate both wells at room temperature for 5 minutes with shaking at 300 rpm.

[0271] (G) Add 50 μL of luciferin generation reagent to each well and shake both wells for 1 minute.

[0272] (H) Incubate both wells at room temperature for 29 minutes.

[0273] (I) Add 100 μL of luciferin detection reagent to both wells and shake them for 1 minute.

[0274] (J) Incubate both wells for 14 minutes.

[0275] (K) Transfer both wells to an assay plate containing a calibration curve (as prepared appropriately in the supplier's protocol).

[0276] (L) Measure luminescence of each well with FLUOstar® Omega "GSHG Assay"

[0277] This example can be followed by the endpoint analysis of Example 5.

[0278] Example 5

[0279] ROS endpoint

[0280] This specific example is based on the ROS-Glo TM The non-lytic variant of the H202 Assay Kit Catalog # G8820 and G8821 and described in the Technical Manual TM391 from Promega.

[0281] (a) Add 75 μL of the sample media collected in Example 1, step 2, and 75 μL of the compound interference control to the assay plate.

[0282] (b) Add 10 wells (100 μL per well) to the assay plate for the standard curve calibration.

[0283] (c) Add 75 μL of freshly prepared ROS-Glo detection solution to all sample wells and compound interference control wells TM If D-cysteine is not present in the reagent, add D-cysteine on top, preferably 0.01 to 20 mM, preferably 0.1 to 10 mM.

[0284] (d) Add 100 μL of ROS-Glo detection solution to the standard curve wells TM .

[0285] (e) Shake the assay plate on a plate shaker set to 300 rpm for 1 minute at room temperature.

[0286] (f) Incubate the plate at room temperature for 20 minutes.

[0287] (g) Measure luminescence of each well using the pre-set "ROS-GloTM H202assay" with FLUOstar® Omega.

[0288] All oscillations during the examples were performed at the stated amounts + / - 10 rpm.

[0289] Room temperature is 18-25°C.

[0290] Example 6

[0291] Figure 7 A schematic showing the experimental protocol used in Example 6.

[0292] In this example, oxidative stress was simulated by adding menadione sodium bisulfite to four samples. The four samples had 10 μΜ, 30 μΜ, 100 μΜ or 300 μΜ menadione sodium bisulfite added to the medium to simulate increasing levels of oxidative stress, respectively. A fifth sample was used as a vehicle control, with 0.3% water added to the medium. The assay was performed on NHBE cells in triplicate.

[0293] The results of Example 6 are shown in Figure 8 .

[0294] The top row shows the results of the assay of the application after 1 hour incubation with menadione sodium bisulfite (left hand panel) and after 2 hours incubation with menadione sodium bisulfite (right hand panel). The right hand panel shows the GSH / GSSG ratio for each sample after 1 hour (dark bar) and 2 hours (light bar). The asterisk ) indicates significance P<0.05 paired t-test.

[0295] In a direct comparison, Figure 8 the bottom row shows the same cell cultures and time points as the top row, but in this case the unmodified assay described herein was used. It can be seen that the results are very similar. This indicates that the application has no detrimental effect on the results.

[0296] Example 7

[0297] Figure 9 A dose effect on the experiment of Example 6 (as shown in Figure 8 ) is shown.

[0298] In the top row of Figure 9 , the same experiment of the application as shown in the top row of Example 6 ( Figure 8 ) was performed. In addition, the ROS endpoint assay of the application (as per Example 5 above) was performed on the same samples at the 1 hour and 2 hour time points (as shown in the bottom row of Figure 9 ). The asterisk Indicates significance P < 0.05 paired t-test. Two asterisks Indicates significance P < 0.01 paired t-test.

[0299] The cells used were NHBE cell lines exposed to water-soluble menadione sodium bisulfite.

[0300] As can be seen from the bottom row of Figure 9 , 30 mM menadione sodium bisulfite increased ROS but did not cause a change in GSH / GSSG. In other words, the level of oxidative stress did not change. However, 100 mM menadione increased ROS and was accompanied by a decrease in GSH / GSSG. This indicates low intensity oxidative stress in the cells. The highest dose of menadione (300 mM) induced the highest ROS and the lowest GSH / GSSG (6-fold decrease). This result was associated with a decrease in cell viability and indicates strong oxidative stress. As shown by the comparison of the 1 hour and 2 hour time points, longer exposure to menadione was associated with more oxidative stress and cytotoxicity (as determined by ATP levels).

[0301] Example 8

[0302] Figure 10A Details are provided regarding the 3D bronchial culture test system used in Figure 11 and Figure 12 , including culture conditions and cell type composition at the time of primary cell differentiation. Figure 10B An enlarged portion of Figure 10A is shown.

[0303] A 3D bronchial culture can be provided by isolating bronchial cells or using commercially available primary cells, culturing them and seeding them on a 3D frame. Suitable cultures include MucilAir TM . MucilAir TM is a cell model of a human airway epithelial culture at an air-liquid interface, available from Epithelix (e.g., here https: / / www.epithelix.com / products / mucilair). In vitro

[0304] Figure 11 ​To illustrate another schematic diagram of how the GSH / GSSG and ROS assays (as detailed in Examples 4 and 5 above) were adapted for 3D bronchial cultures. Three 3D cultures were used in this experiment. One was supplemented with 100 μM menadione sodium bisulfite to simulate oxidative stress, a second with 300 μM menadione sodium bisulfite, and a third was a mediator control in which only culture medium and water were added. The bottom of the figure shows how samples were collected from the 3D bronchial cultures to be evaluated by the modified GSH / GSSG-Glo and ROS-Glo assays. The GSH / GSSG-Glo assay was performed on samples from cell cultures within the culture insert, and the ROS-Glo assay was performed on the culture medium outside the substrate. Triple copies were performed.

[0305] Figure 12 It shows in Figure 11 Results of experiments conducted. (Significance levels are shown at the bottom of the figure). The GSH / GSSG ratio used in the adjusted GSH / GSSG and ROS determination media controls according to the invention is reproducible and similar to the GSH / GSSG ratio observed under 2D conditions, indicating that the protocol is also effective for 3D cell cultures. Lower doses of menadione sodium bisulfite significantly increased ROS but had no effect on GSH / GSSG. Higher doses further decreased GSH and GSH / GSSG but had no further effect on ROS and GSSG.

[0306] Example 9

[0307] Figure 13 The results of the assays performed according to Examples 4 and 5 to test the applicability of the invention to different cell lines are shown.

[0308] In this experiment, the A549 lung cell line was exposed to the lipid-soluble menadione (vitamin K3) to simulate oxidative stress. This induced changes in the lung cell line compared to those from... Figure 9 Similar results were observed in NHBE cell lines exposed to water-soluble menadione sodium bisulfite, where ROS increased in a dose-dependent manner and GSH / GSSG decreased in a dose-dependent manner, affecting cell viability at both highest concentrations.

[0309] asterisk ( The significance level (P < 0.05) is indicated by a paired t-test. Two asterisks (...) The significance level is indicated by a paired t-test (P < 0.01).

[0310] Example 10

[0311] Figure 14To show a schematic of the experimental protocol, the results obtained in 3D bronchial cultures by using liposoluble menadione for the determination of the application as performed in Example 4 and Example 5. Figure 15

[0312] 3D bronchial cultures according to Example 8 were used.

[0313] Figure 15 It is shown that the dose-dependent decrease of GSH and GSH / GSSG and the increase of GSSG and ROS after exposure of 3D bronchial cultures to liposoluble menadione had no impact on cell viability, in contrast to the results from the use of menadione sodium bisulfite. Figure 11 This indicates that the application can capture different effects on glutathione levels induced by different chemical forms of menadione.

[0314] Overall results

[0315] • The disclosed method solves known problems with the described improved kit.

[0316] • The method saves time and costs for tissue handling (1 tissue instead of 3 per case = 66% cost reduction).

[0317] • The cost reduction is particularly significant for 3D cell culture work, where costs are reduced by about two thirds.

[0318] • The method of the application shows a time-dependent effect.

[0319] • This overall method improves the biological relevance, as 3 oxidative stress readouts originate from the same sample, thus eliminating biological (inter-tissue) variability, while also minimizing technical variability.

[0320] • It provides a scientifically comprehensive characterization of oxidative stress, which by definition cannot be achieved by assessing only a single biomarker.

[0321] • The disclosed method is compatible with high-throughput assessment and is less susceptible to compound interference.

[0322] ​Any publication cited or described in this document provides information as of the date of submission of the instant application. The statements herein are not to be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior application. All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the application will be apparent to those skilled in the art without departing from the scope and spirit of the application. Although the application has been described in connection with specific preferred embodiments, it should be understood that the application as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes of carrying out the application that are obvious to those skilled in engineering, cell biology and molecular biology or related fields are intended to be within the scope of the following claims.

Claims

1. A method of measuring oxidative stress in a cell or tissue sample, the method comprising the steps of: (i) treating the cell or tissue sample in a culture medium; (ii) collecting a portion of the culture medium; (iii) measuring the amount of glutathione in the cell or tissue sample; and (iv) measuring the amount of reactive oxygen species (ROS) in the portion of culture medium collected in step (ii); wherein step (iii) is performed immediately after steps (i) and (ii); and wherein step (iv) is performed simultaneously with step (iii) or within 6 hours of the completion of step (iii) if the collected portion of culture medium is stored at about 0-8°C prior to performing step (iv), including assay time.

2. The method of claim 1, wherein the amount of ROS and / or the amount of glutathione is measured using luminescence, preferably wherein the amount of ROS and / or the amount of glutathione is measured quantitatively or qualitatively or relatively.

4. The method of any preceding claim, wherein the treatment in step (i) is for a duration of about 6 hours or less.

3. The method of any preceding claim, wherein the culture medium of step (i) comprises a hydrogen peroxide (H202) substrate, suitably a ROS-Glo TM H202 substrate.

5. The method of any preceding claim, wherein the amount of glutathione measured in step (iii) is the total of reduced glutathione (GSH) and oxidized GSH (GSSG).

6. The method of any preceding claim, wherein the GSH / GSSG ratio is calculated in step (iii), preferably wherein the GSH / GSSG ratio is calculated using relative luminescence units (RLU).

7. The method of any preceding claim, wherein the amount of GSH is measured in step (iii) using a reducing agent, preferably a reducing agent capable of reducing disulfide bonds, preferably wherein the reducing agent is dithiothreitol (DTT), dithioerythritol, dithionite, sodium borohydride, potassium borohydride, tris(2-carboxyethyl)phosphine (TCEP), triphenylphosphine, or tributylphosphine. (d) dividing each well containing a lysed cell or tissue sample into two wells in the assay plate; 8. The method of any preceding claim, wherein step (iii) comprises at least the following steps: - (a) placing the cell or tissue sample in a well plate, adding GSH-Glo TM reagent to each well and shaking at about 300 rpm for about 5 minutes at room temperature to lyse the cells; (b) Add GSH-Glo to standard curve wells in assay plate TM reagents; (c) adding GSH-Glo to the reductant in the assay plate and to one or more aqueous vehicle background control wells TM reagent, if D-cysteine is not present in the reagent, preferably 0.01 to 20 mM, preferably 0.1 to 10 mM; (e) adding a reducing agent to one of each pair of sample wells and to at least one reducing agent background control well for measuring total glutathione; (f) adding water to the remaining sample wells without reducing agent and to the one or more water vehicle background control wells for measuring reduced GSH; (g) shaking all wells at about 300 rpm for about 1 minute at room temperature; (h) incubating all wells at room temperature; (i) adding a reagent capable of revealing signal from a luciferin-luciferase system to the sample wells, to at least one reducing agent well and one or more water vehicle background control wells, and to the standard curve wells; (j) shaking all wells at about 300 rpm for about 1 minute at room temperature; (k) incubating all wells at room temperature; and (l) measuring luminescence. ​ 9. The method of claim 8, wherein the reducing agent is capable of reducing disulfide bonds, preferably wherein the reducing agent is dithiothreitol (DTT), dithioerythritol, dithionite, sodium borohydride, potassium borohydride, tris(2-carboxyethyl)phosphine (TCEP), triphenylphosphine, or tributylphosphine.

10. The method of any one of claims 8-9, wherein step (I) is performed using a luminometer plate reader.

11. The method according to any one of claims 1-7, wherein step (iii) comprises at least the following steps: - (A) removing cell supernatant from the sample; (B) adding passive lysis buffer to the sample and shaking at about 300 rpm for about 5 minutes; (C) dividing the sample equally into two wells; (D) adding luciferin-NT to one well to quantify total glutathione; (E) adding luciferin-NT and N-ethylmaleimide (NEM) to the other well to quantify oxidized glutathione (GSSG); (F) incubating both wells at room temperature with shaking at about 300 rpm for about 5 minutes; (G) adding luciferase generating reagent to each well; shaking for about 1 minute; (H) incubating both wells at room temperature; (I) adding reagents to both wells capable of revealing signal from the luciferin-luciferase system and shaking for about 1 minute; (J) incubating both wells for about 14 minutes; (K) transferring the sample wells to an assay plate containing a calibration curve; and (L) measuring luminescence.

12. The method of claim 11, wherein step (L) is performed using a luminometer plate reader.

13. The method of any preceding claim, wherein step (iv) comprises at least the following steps: - (h) adding a media only control sample and an equivalent amount of compound interference control to the assay plate; (i) adding a standard curve to at least 10 wells of the assay plate; (j) adding reagents to all wells capable of revealing signal from the luciferin-luciferase system, adding D-cysteine if not already present in the reagents, preferably 0.01 to 20 mM, preferably 0.1 to 10 mM; (k) shaking the assay plate at about 300 rpm for about 1 minute at room temperature; (I) incubating the assay plate at about room temperature for about 20 minutes; (m) measuring luminescence.

14. A kit comprising GSH-Glo from Promega TM assay kit or GSH / GSSG-Glo TM kit, plus reducing reagents and instructions.

15. The kit of claim 19, further comprising ROS-Glo from Promega TM H2O2assay.

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