Rapid Quantification of Oxidized and Reduced Forms of Glutathione Using Ortho -phthalaldehyde in Cultured Mammalian Cells In Vitro

Summary

Quantification of both oxidized and reduced forms of glutathione (GSSG and GSH, respectively) has been achieved through the use of Ortho-phthalaldehyde (OPA). OPA becomes highly fluorescent once conjugated to GSH but is unable to conjugate GSSG until reduced. Here, we describe a multiparametric assay to quantify both using protein quantification for normalization.

Abstract

Glutathione has long been considered a key biomarker for determining the antioxidant response of the cell. Hence, it is a primary marker for reactive oxygen species studies. The method utilizes Ortho-phthalaldehyde (OPA) to quantify the cellular concentration of glutathione(s). OPA conjugates with reduced glutathione (GSH) via sulfhydryl binding to subsequently form an isoindole, resulting in a highly fluorescent conjugate. To attain an accurate result of both oxidized glutathione (GSSG) and GSH, a combination of masking agents and reducing agents, which have been implemented in this protocol, are required. Treatments may also impact cellular viability. Hence, normalization via protein assay is presented in this multiparametric assay. The assay demonstrates a pseudo-linear detection range of 0.234 - 30µM (R²=0.9932±0.007 (N=12)) specific to GSH. The proposed assay also allows for the determination of oxidized glutathione with the addition of the masking agent N-ethylmaleimide to bind reduced glutathione, and the reducing agent tris(2-carboxyethyl) phosphine is introduced to cleave the disulfide bond in GSSG to produce two molecules of GSH. The assay is used in combination with a validated bicinchoninic acid assay for protein quantification and an adenylate kinase assay for cytotoxicity assessment.

Introduction

Reactive oxygen species (ROS) are a primary inducer of oxidative stress; oxidative stress has been well established in the generation of DNA mutations, cellular aging/ death, various cancers, diabetes, neurological diseases (such as Parkinson's and Alzheimers), and several other life-debilitating conditions^1,2,3,4,5. A key defense against ROS are thiolic, non-enzymatic antioxidants, which are capable of reducing oxidants or radicals by acting as proton donors^6,7. Glutathione (GSH) and cysteine are the two most prevalent thiols found in mammals⁸, while various other low molecular weight thiols exist (such as ergothioneine), GSH and cysteine are the most commonly measured non-enzymatic antioxidants found in literature^9,10,11 and hold greatest relevance for combatting ROS^8,12,13,14.

When GSH is utilized as an antioxidant, two molecules of GSH are covalently linked together via a disulfide bond to make glutathione disulfide (GSSG). Depletion of GSH is often used as an indicator of oxidative stress^15,16. This assessment can also be combined with the detection of GSSG, although increases in GSSG in cells are often limited by active export processes since GSSG can be relatively reactive in cells, leading to disulfide bond formation with other protein thiols¹⁶.

Traditional methods for measuring GSH and GSSG are not simple processes and require numerous steps, including cellular extraction using lytic reagents^17,18. The protocol outlined here simplifies these methods and allows the accurate measurement of non-enzymatic thiols and normalization using the cellular protein content or adenylate kinase release. In addition, it is possible to measure cellular viability prior to GSH/GSSG extraction. Several methods have previously attempted to target and quantify reduced and oxidized non-enzymatic thiols efficiently; methods including the use of HPLC^19,20,21, plate assay (biochemical)^22,23,24,25, and which use common reagents for thiol conjugation, such as 5,5-dithio-bis-(2-nitrobenzoic acid) (DTNB/ Ellman's reagent)¹⁹, Monochlorobimane (mBCI)^26,27,28. Several companies have also prepared proprietary kits for the detection of glutathione; however, they do not publish reagent incompatibilities, which presents issues dependent on the treatments used²⁹.

This protocol outlines a multiparametric assay that detects reduced thiols (such as GSH) via ortho-phthalaldehyde (OPA) conjugation to produce a fluorescent signal detectable at 340/450 Ex/Em, respectively. This assay facilitates the detection of both GSH and GSSG simultaneously (in plate), through the use of masking agents (N-ethylmaleimide) and GSSG reducing agents (tris(2-carboxyethyl) phosphine). This multi-biomarker protocol also provides an opportunity during the cellular lysing stage to quantify proteins via bicinchoninic acid assay for the normalization of samples on completion of the final measurement or via an adenylate kinase assay from the cell media. This assay can be performed utilizing several reagents readily available in most laboratories and only requires a few additional uncommon chemicals to perform. The process is simple, accessible, and can be performed without laborious stages in less than 2 h.

In this protocol, various nanomaterials were chosen that were either previously shown to induce ROS or suspected to induce oxidative stress^30,31. A concentration range was explored to see the effects of exposure of these nanomaterials on various cell lines and the effectiveness of the assay in quantifying antioxidant thiols.

Protocol

NOTE: The following protocol has been designed with the capacity to be utilized in conjunction with a bicinchoninic acid (BCA) protein assay and an adenylate kinase (AK) assay to normalize samples to treatments. Ensure the operator is wearing appropriate attire and necessary safety equipment, such as a Howie lab coat, nitrile gloves, and class I safety glasses, throughout the preparation and use of materials. The protocol is divided into several stages.

1. Stock and working solutions preparations

1. Prepare stock solutions of 100 mM GSH standard in 1 mM HCl (prepared from 37% HCl in double distilled water (ddH₂O)).
   NOTE: If diluting from highly concentrated acid, such as 37% HCl, ensure the correct process of adding acid to water in a class I fume hood.
2. Prepare a stock of 22.35 mM OPA in absolute ethanol. Perform this step in a class I fume hood. Prepare 25 mM N-ethylmaleimide (NEM) in ddH₂O. Perform this step in a class I fume hood.
   NOTE: These three solutions can be stored at -20 °C for up to 3 months.
3. Prepare 0.01 M Tris(2-carboxyethyl) phosphine (TCEP) to a total volume of 500 µL, required for 100 wells. Prepare 100 µL of 1 mM GSH standard, diluted from 100 mM stock using ddH₂O.
4. Either use immunoprecipitation (IP) lysis buffer or the following formulation: 394 mg of Tris-HCl (final concentration 25 mM), 877 mg of NaCl (final concentration 150 mM), 29 mg of EDTA (final concentration 1 mM), either 1 mL of 100% NP-40 or IGEPAL CA-630 (final concentration of 1% V/V), 5 mL of glycerol (final concentration 5% V/V), 84 mL of ddH₂O. Mix components via gentle stirring and adjust pH to 7.4. Decant into a 100 mL volumetric flask and add the remaining volume of ddH₂O to reach a final volume of 100 mL. Sterile filter through a 0.22 µm filter and store at 2-8 °C for up to 6 months.
   NOTE: Lysing solutions do act as a contaminant/interferent in this assay; hence, the above formulations are specified.
5. Prepare 1 L of 0.1 M phosphate-buffered saline supplemented with ethylenediamine tetraacetic acid (PBS-EDTA) at 3 different pH values, specifically 7.2, 8.5, and 9.0. Use a commercial 0.1 M PBS buffer supplemented with 1 mM EDTA (292.24 mg/L) or the following 10x (1 L) formulation: 80 g NaCl, 2.0 g KCl, 14.4 g Na₂HPO₄, 2.4 g KH₂PO₄, 800 mL of ddH₂O. Add and mix; top up to 1 L. Autoclave the solution for sterility and store it for 12 months at room temperature. From the 10x stock solution, make a working solution of PBS and supplement it with EDTA (concentration as stated previously).
   NOTE: pH is of critical importance; ensure pH is accurate before commencing protocol.
6. Perform 1:2 serial dilution of 1 mM GSH using ddH₂O (1 mM, 500 µM, 250 µM, 125 µM, 62.5 µM, 31.25 µM, 15.625 µM, and 7.8125 µM). Add 10 µL of each concentration to standard wells (performed in duplicate). Samples will be further diluted in lysis buffer; hence, concentrations will be 1/5^th of their original concentration afterward (200, 100, 50, 25, 12.5, 6.25, 3.125, 1.5625 µM).
   NOTE: Final concentrations for calibration will be 30 µm,15 µm,7.5 µm, 3.75 µm, 1.875 µm, 937.5 nm, 468.8 nm, and 234.4 nm.

2. Assay preparation

NOTE: This protocol uses human cell lines HepG2, A549, and J774, which were purchased commercially from ATCC. These cell lines were utilized under the approved guidelines outlined by the animal and tissue culture acts and regulations of the University.

1. At 24 h prior to assay commencement, seed cells at concentrations shown in Table 1; however, depending on the cell line/ type and treatment used, adjust density as needed. Grow cells in complete growth medium (Eagles modified essentials medium (EMEM) with 10% Heat-inactivated fetal calf serum (HIFS), 1% Penstrep (10,000U/mL penicillin/ 10mg/mL streptomycin), and 1% non-essential amino acids.
2. Seed cells using conventional cell seeding methods³². Seed cells in fully black plates if not using post-assay microscopy. Assess cells for confluency and general health in the T75 flask prior to use via microscopy.
3. In a clean, sterile, class II biological safety cabinet and following a strict aseptic technique, discard cell media, gently wash cells with ~15 mL of sterile, room temperature (RT) PBS, and discard.
4. To the cell flask, add 5 mL of sterile 1x trypsin, gently rock to ensure coverage of the cell monolayer, and place into an incubator at 37 °C for 5 min to facilitate cell detachment.
5. Stop trypsinization by addition of growth medium containing heat-inactivated fetal calf serum (10%), approximately 10 mL.
6. Transfer cells to a 50 mL centrifuge tube and pellet at 200 x g for 5 min. Discard the media and replace it with 5 mL of the same media. Resuspend cells in the tube until homogenous and no clumping can be observed.
7. Remove 20 µL of cell suspension and place in a hemocytometer for counting. Once the number of cells required has been calculated, perform a dilution to make a solution with the correct cell density.
8. Pipette media containing cells in 96-well plates (350 µL capacity), with a maximum volume of 200 µL per well. Place cells in a 37 °C incubator with 5% CO₂ for 24 h to adhere to the plate surface.
9. To assess both total glutathione and GSSG, treat samples for both conditions. Seed and treat 6 wells to provide 2 sets each of GSH and GSSG with 3 technical repeats (Figure 1 demonstrates layout).
10. Ensure all reagents are appropriately prepared prior to the commencement of the assay; ensure all buffer components and reagents are at RT before commencing construction of buffers or use in the assay, with the exception of pH 7.4 PBS (without EDTA), which should be kept on ice/ refrigerated until use.
    NOTE: It is of critical importance that bubble formation be limited to a minimum to allow desired reactions to occur within the assay and allow accurate quantification via plate reader.

Cell line	Seeding density (96-well plate)
HepG2	10,000 cells / well
A549	5,000 cells / well
J774	10,000 cells / well

Table 1: Suggested seeding densities for chosen cell lines. Demonstrated are different seeding densities for three different cell lines used in the represented data, specifically A549, J774, and HepG2.

Figure 1: Proposed layout for seeding of 96 well plates for the simultaneous determination of total Glutathione and Glutathione disulfide. Wells for calibration and controls are also demonstrated. Wells that are not utilized are represented with a cross. Please click here to view a larger version of this figure.

3. Nanomaterial treatment

1. Weigh nanomaterials (specifically ZnO, TiO₂, CuO, and Ag) in a µg capable weight scale. Perform a calculation to attain an initial concentration of 1 mg/mL per nanomaterial.
2. Transfer nanomaterial solutions to a sonicator and sonicate for 16 min using a sonication bath (38 W) to produce a homogenous solution. Make a series of dilutions for each nanomaterial at 125, 62.5, 31.25, 15.625 µg/mL.
3. Remove cells from the incubator and lightly wash with RT PBS. After ensuring all PBS has been removed, add 100 µL of treatments to the plate, with controls (culture media without HIFS). After applying treatment to cells, incubate with the nanomaterials in a 37^{°C incubator (5% CO}₂) for 4 h; afterwards, use the protocol below to quantify GSH: GSSG, protein concentration, and AK release.

4. Assay protocol

1. After exposure to treatment (Figure 2A), assess viability via adenylate kinase (AK) activity (optional). Use a commercial kit for this assay following the manufacturer's instructions.
2. Spin plates in a centrifuge at 200 x g for 5 min to lightly pellet treatment and cell debris (Figure 2B).
3. Remove 20 µL of media supernatant gently from each sample and control well (as specified above) and pipette into an adjacent 96-well white plate in the same layout format (Figure 2C).
4. Add 100 µL of working solution from the AK kit to each well, shield the plate from light, and leave to develop for 10 min at room temperature. Record luminescence using a plate reader at 1000 counts/s.
5. For GSH standards, add 40 µL of total glutathione buffer to each well (Figure 2D; optional, required for quantification).
6. Aspirate remaining media from the plate and wash 3x with ice-cold 0.1 M PBS, pH 7.2, discarding each wash (Figure 2E) with the exception of standards. Leave the final wash in the plate until the GSH calibration has been loaded into the plate.
7. Add 10 µL of each glutathione concentration and blank (ddH₂O) into wells in triplicate.
8. Remove the final PBS wash and add the mixtures to each well as per Table 2 for desired target quantification (Figure 2E). Calculate required volumes prior to commencing this step due to time-sensitive loss of activity with complete buffers. Ensure these reagent mixes are made before starting the assay process, but do not allow them to sit for more than 30 min before use.
9. Place on an orbital plate shaker and allow the plate to shake at 300 RPM for 2 min (Figure 2F).
10. Remove from the shaker and add 5 µL of 0.01M TCEP solution to each well, excluding the NEM control well. Return the plate to the shaker and incubate for 10 min (Figure 2F).
11. Transfer plate to centrifuge and spin at 200 x g for 5 min. Transfer 25 µL from each sample well to another 96-well plate (clear); this will be utilized for protein concentration (see step 4.15 ; Figure 2G). Do not transfer standards or assay controls.
12. Ensure the final volume for each well is 30 µL; remove volume from controls and standards to meet this requirement (Figure 2H).
13. Add 170 µL of working OPA solution to each well, shield plate from light and place on shaker for 15 min (Figure 2I).
14. Read fluorescence using a plate reader at Ex340/Em450 (Figure 2J). Ensure no bubbles are present during the measurement stage; they will have a detrimental impact on both the reaction and the quantification via plate reader.
15. To quantify the protein content of lysed cells, use a commercial BCA assay kit. Transfer samples taken from step 4.12 to a new 96-well plate (clear) at 25 µL per well.
16. Use a bovine serum albumin (BSA) standard diluted in IP lysis buffer and add to the plate in triplicate at 25 µL per well. The exact concentrations are defined in the BCA kit protocol.
17. Prepare a working solution consisting of 50:1 ratio of reagents A and B from the BCA kit and add 200 µL to each well containing sample, standard, and control. Shield plates from light and incubate at 37^{°C for 30 min} (Figure 2K).
18. Remove samples from the incubator, allow to equilibrate at room temperature for 5 min, then read absorbance via plate reader at 562 nm (Figure 2L).

Total glutathione concentration lysis reagent mix
Component	Volume
Lysis buffer	50µL
Total volume / well	50µL
Oxidised glutathione concentration lysis reagent mix
Component	Volume
Lysis buffer	49.5µL
NEM (25mM)	0.5µL
Total volume / well	50µL
USE BOTH SOLUTIONS WITHIN 30 MINUTES OF MIXTURE COMPOSITION
OPA detection solution component	Volume
OPA 3mg/mL	5µL
PBS (pH 9.0)	165µL
Total volume / well	170µL

Table 2: Necessary volumes of reagents for performing the protocol. Volumes required per well for the determination of total glutathione, glutathione disulfide, and working reagent required. Ensure required volumes are calculated, and an excess is included to account for volume loss through transfer.

Figure 2: Schematic representation of the protocol. (A) Initial seeding, incubation and treatment of cells. (B) Centrifugation to separate media from suspended solids. (C) Media transfer for Adenylate kinase assay. (D) Addition of glutathione concentrations for calibration range. (E) Washing stages and lysing reagent addition. (F) Buffer addition and tris(2-carboxyethyl) phosphine addition with shaking step. (G) Centrifugation of lysed cells for media removal for protein analysis. (H) Media removal to equalize volume across the plate. (I) Addition of ortho-phthalaldehyde working solution with shaking incubation. (J) Measurement of ortho-phthalaldehyde fluorescence via plate reader. (K) Incubation stages for Bicinchoninic acid assay for protein determination. (L) Measurement of protein concentration, allowing normalization of glutathione: glutathione disulfide values. Please click here to view a larger version of this figure.

Representative Results

Following this protocol, A549 and J774 cell lines were seeded at densities of 5,000 cells/ well and 10,000 cells/well, respectively and cultured at 37 °C in 5% CO₂ for 48 h. The AK analysis after nanomaterials treatment is shown in Supplementary Table 1, and the protein concentration is shown in Supplementary Table 2.

Calibration graph
Shown in Figure 3 are three calibrations using the stated concentration range (0.234 - 30 µM final concentration) from three separate plates from three different cell types (though should not impact calibration) on three different, non-consecutive, days. While 3 samples are shown, an N of 12 was observed, and demonstrated similar linear regressions with an average R² value of 0.9932 ± 0.007.

Figure 3: Glutathione calibration graphs for assay. Three calibration graphs from separate in-plate glutathione calibration ranges, each performed a week apart; error bars ± SD (n=3, N=12) n=technical replicates, N=Biological replicates. Please click here to view a larger version of this figure.

Sample results
HepG2, A549, and J774 cells were utilized in the assessment of various nanomaterials suspected of inducing changes to cellular mechanisms via oxidative stress. The detection and quantification protocol described were utilized.

The data received from the 3 measurements (AK, BCA, and GSH/GSSG) was handled as follows. The AK and BCA assay was implemented for normalization; the AK assay, using the recommended kit, will give the fastest, simplest data for the amount of AK released into the cell media. An increase in AK values is expected for increasing cell death. Hence, a -ve (Alive) and +ve (Dead) control is required. This will allow for normalization based on percentage.

The BCA assay is a longer process but will allow for quantifiable results to be acquired via protein quantification (mg/mL). This does not require a -ve or +ve control as in the AK but will still require a general -ve control (untreated cells) to allow for the normalization of values to be achieved.

In this representative results section, it was found that the treatment (nanomaterials) had the potential to cause interference with the AK assay. Hence, all normalization was performed using the BCA data. Therefore, information is presented as the concentration of detected species (GSH or GSH+GSSG (however, a subtraction of GSH concentration from total GSH+GSSG concentration is performed to get GSSG concentration) per mg/mL of protein (via BCA assay). If desired, this can then be converted into a ratio to assess the change in GSH: GSSG from the desired treatment.

Shown in Figure 4 is the GSH: GSSG ratio data from three different cell lines (A549, J774, and HepG2) acquired using the OPA protocol and normalized to protein expression via BCA (µg/mL), further data specifying additional GSH and GSSG values can be found in Supplementary Figure 1.

Figure 4: Glutathione: Glutathione disulfide ratiofrom performing the assay. Shown are the glutathione: Glutathione disulfide ratios of 3 cell lines, namely (A) A549, (B) J774, and (C) HepG2. Cells were incubated with treatments (various nanomaterials in serum-free media) for 4 h. Cells were processed using this protocol to quantify changes in glutathione and glutathione disulfide and normalized via protein quantification, error bars ± SE (n=3, N=3) Please click here to view a larger version of this figure.

The plate also contains a series of controls to ensure the assay has run correctly. NEM is added as an individual component to demonstrate a lack of interaction with OPA detection media. The calibration standard demonstrates a linear increase with GSH concentration, which demonstrates the effective capacity for the OPA detection reagent to effectively bind to increasing concentrations of GSH.

It must be noted that this assay specifically targets free sulfhydryl groups commonly found in thiols (such as GSH, which are commonly considered antioxidants). One potential interaction is the binding of OPA to protein thiols, which would result in inaccurate data gathering. Hence, the BCA assay is a crucial stage to normalize data to protein and allow accurate reflection of free GSH.

Supplementary Figure 1: Figures demonstrating glutathione, glutathione disulfide, and glutathione: glutathione disulfide ratio from 3 cell lines, namely, (A) A549, (B) J774, and (C) HepG2. Cells were incubated with treatments (various nanomaterials in serum-free media) for 4 h. Cells were processed using this protocol to quantify changes in glutathione and glutathione disulfide and normalized via protein quantification, error bars ± SE (n=3, N=3) Please click here to download this File.

Supplementary Table 1: Metadata of adenylate kinase values for A549 and J774 cells. Please click here to download this File.

Supplementary Table 2: Metadata of Bicinchoninic acid values with calibration for A549, J774, and HepG2 cells. Please click here to download this File.

Discussion

As stated, the need to understand cellular redox, monitor states of oxidative stress, and the antioxidant response has always been crucial in understanding and preventing a myriad of diseases, such as cancers and neurodegeneration^33,34. Demonstrated here is a means to improve upon the translational landscape by increasing the accessibility of accurate GSH: GSSG detection with fast, minimal preparation.

This protocol demonstrates a multiparametric sequence of assays for the determination of intracellular glutathione/ thiol species (reduced and oxidized), with 2 means of normalization via BCA protein assay and/or AK assay. This assay can also be modified to detect various other markers through the initial mediator extraction step and can be simplified in a manner that will simply give an oxidized/reduced thiol ratio with the exclusion of the calibration range.

When considering the evaluation of the analytes, both mBCI and OPA were explored and compared for use. While mBCl initially demonstrated good signal potential, significant limitations in use were discovered. Primarily, the use of live cells demonstrates the best use of mBCl; however, after cell lysis, the signal was found to be quenched and is generally diminished in a multiwell format compared to OPA³⁵. Another issue is the measurement of GSSG via mBCl, literature is sparse regarding this, and through protocol optimization/ exploration, accurate detection of GSSG through mBCl was not achieved.

We have demonstrated that OPA assay presents significantly reliable calibration ranges, with an R² average of 0.9932 ± 0.007 (N=12) across a 0.234 - 30 µM GSH concentration range. This range was chosen due to previous reference ranges found in the literature³⁵. It is theoretically possible to detect glutathione outside of these ranges but will require modification to the concentration of reagents, incubation time, and potentially the equipment utilized in detection. It must be noted that each plate requires its own standard range for quantification; the slightest variance in time between plates performed on different days can have a significant effect on the values obtained during measurement.

Achieving accurate and reliable data from this protocol is dependent on several crucial steps being strictly adhered to. When constructing the various buffers required in the protocol, it is crucial that pH is accurate. Hence, buffers requiring a pH of 9 should have no deviation beyond ± 0.1 of this value. This is due to the potential for buffer components to precipitate out of the solution in the wrong pH; following this protocol exactly will prevent this issue.

Complete removal of treatment and accurate washing before lysing are also critical to prevent artifacts and inaccurate data acquisition during the plate reading stage. Once cells have been lysed (step 4.8), removal of treatment is not possible, and the plate will not be salvageable. Due to the volumes of buffers/reagents being added throughout the protocol varying between samples and standards, it is critical that the user is aware of the varied volumes, in step 4.12 and 4.13. The assay operator is also made aware of these varied volumes and instructed to ensure all volumes are the same to allow accurate measurement to be achieved. As the volumes between the samples and standards are not visibly significant, it can be an easy mistake to make with regard to having an excess solution in the sample well.

There are limitations to this protocol that rely on crucial steps, which are critical to acquiring accurate and reliable data. The users performing this assay need to possess a reasonable level of laboratory skills to prevent undesired problems, such as bubble formation. Bubble formation has a drastic impact on both the capacity for reactions to occur within the microplate and the measurement of fluorescence. The lysing agent used in this protocol contains a detergent, which presents difficulty to a novice researcher who may struggle to prevent bubble formation. Immediate centrifugation may salvage this error. The protocol is also potentially limited regarding cell type; cell lines A549, J774, and HepG2 were utilized to both optimize and produce data for this protocol. Other cell lines may require different seeding densities and optimization of the protocol to get accurate data.

This protocol offers numerous advantages over several existing assays. While the detection of thiols using Phthalaldehyde is not a novel concept, utilization in a combined assay format such as this, in a microplate, with limited required materials and equipment, offers great potential for all labs to access this protocol. Most Thiol/ GSH kits from commercial suppliers do not disclose the composition of their reagents. Hence, it can be difficult to foresee the potential for incompatibilities/ interference. Here, we present each component of all the utilized reagents to limit that potential.

This protocol is also performed rather rapidly upon completion of the initial treatment period. Accounting for user processing between incubation stages, the thiol quantification aspect of this protocol can be performed in under 1 h. Samples are simultaneously lysed and bound to prevent auto-oxidation of samples, which is optimal for these reaction species. While not specified in the protocol, samples can technically be lysed in a plate and sealed, allowing them to be frozen for future analysis. However, this alteration to the protocol has not been explored.

Materials

Name	Company	Catalog Number	Comments
0.22µm filter (optional-For lysis buffer)	Fisher scientific	12561259
100mL volumetric flask	Fisher scientific	15290866
1L Volumetric flask	Fisher scientific	15230876
250mL beaker (optional-For lysis buffer)	Fisher scientific	15409083
8-Channel micropipette (20-200µL)	SLS	FA10011D2
8-Channel micropipette (2-20µL)	SLS	B2B06492
96 well plates - black with clear bottom, TC treated	Fisher scientific	10000631	Preferred plate for seeding and fluoresence, use TC treated clear if unavailable
96 well plates - clear (TC treated and untreated)	Fisher scientific	10141161	If black plates with clear bottom is not available/ suitable use TC treated clear
96 well plates - white, Not TC treated	Fisher scientific	11457009
A549 (lung carcinoma) cell line	ATCC	CCL-185
Absolute ethanol	Merck (Sigma-Aldrich)	1.08543
Aluminium foil	Fisher scientific	11779408	For protecting plates from light
BCA Assay Kit	Thermo	23225
Benchtop Centrifuge (with 96 plate rotor)	Eppendorf	5804
Ethylenediaminetetraacetic acid (EDTA)	Merck (Sigma-Aldrich)	E9884
Glutathione (GSH)	Merck (Sigma-Aldrich)	G6013
Glutathione disulfide (GSSG)	Merck (Sigma-Aldrich)	G4501
Glycerol	Merck (Sigma-Aldrich)	G5516
HCl, 37%	Merck (Sigma-Aldrich)	258148	Dilute to 1mM for GSH stock, pH adjustment also
HepG2 (Hepatocarcinoma) cell line	ATCC	HB-8065
IGEPAL CA-630	Merck (Sigma-Aldrich)	18896	Use either IGEPAL CA-630 or NP-40 for solution, not both
IP lysis buffer	Fisher scientific	11825135
J774 (monocyte, macrophage) cell line	ATCC	TIB-67
KCl	Merck (Sigma-Aldrich)	P3911
KH2PO4	Merck (Sigma-Aldrich)	P0662
Micropipette (20-200µL)	SLS	B2B06482
Micropipette (2-20µL)	SLS	B2B06478
Microplate shaker	VWR	444-0041
Na2HPO4	Merck (Sigma-Aldrich)	S9763
NaCl	Merck (Sigma-Aldrich)	S9888
NaOH, 10M	Merck (Sigma-Aldrich)	72068	For pH adjustment only
N-Ethylmaleimide (NEM)	Merck (Sigma-Aldrich)	E3876
NP-40	Merck (Sigma-Aldrich)	492016	Use either IGEPAL CA-630 or NP-40 for solution, not both. NP-40 alternative suggested
Ortho -Phthaldialdehyde (OPA)	Merck (Sigma-Aldrich)	P1378
PBS 0.1M	Merck (Sigma-Aldrich)	P2272	PBS can either be acquired pre-made or made in house, see notes
Plate reader (with fluoresence capacity)	Tecan	SPARK
Stir bar (optional-For lysis buffer)	Fisher scientific	16265731
Toxilight bioassay kit (AK assay)	Lonza	LT17-217
Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) 0.5M  in H2O	Alfa Aesar	H51864	Can also be purchased crystalised and suspended
TRIS-HCl	Merck (Sigma-Aldrich)	93363
X100 phosphatase and protease cocktail	Fisher scientific	10025743