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

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.

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).

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.