GSH/GSSG Assay Kit

Reagent Preparation

1. Enzyme Solution: add 1 mL Buffer Solution, dissolve completely, mix gently, and aliquot to avoid repeated freeze-thaw cycles.

2. Coenzyme Solution: add 1 mL Buffer Solution to the Coenzyme and dissolve completely. Prepare immediately before use.

3. Substrate Solution: add 1 mL Buffer Solution to Substrate (DTNB), dissolve completely, and protect from light.

4. GSH standard stock: add 1 mL Extraction Buffer to Standard GSH and dissolve completely to obtain a 0.4 mM GSH stock solution.

5. GSSG standard stock: add 1 mL Extraction Buffer to Standard GSSG and dissolve completely to obtain a 0.2 mM GSSG stock solution (equivalent to 0.4 mM GSH equivalents).

6. Masking Solution and Quenching Solution: add the volume of Extraction Buffer indicated in the manual to each vial and dissolve completely. Equilibrate to room temperature before use.

Sample Preparation

1. Plasma, serum, or urine: mix the sample with an equal volume of Extraction Buffer, centrifuge at 10,000 × g for 10 min at 4°C, and collect the supernatant.

2. Cells: collect approximately 1 × 10⁶ cells, wash with PBS, add 100-200 μL Extraction Buffer, and lyse thoroughly. Centrifuge at 10,000 × g for 10 min at 4°C and collect the supernatant.

3. Tissue: weigh 10-30 mg tissue, add 100 μL Extraction Buffer per 10 mg tissue, homogenize thoroughly on ice, centrifuge at 10,000 × g for 10 min at 4°C, and collect the supernatant.

Note: Keep samples on ice and complete the assay as soon as possible. For storage, aliquot at -80°C and avoid repeated freeze-thaw cycles.

Standard Curves

1. Dilute the GSH stock with Extraction Buffer to prepare 0, 2, 4, 8, 12, 16, and 20 μM GSH standards.

2. Dilute the GSSG stock with Extraction Buffer to prepare 0, 1, 2, 4, 6, 8, and 10 μM GSSG standards.

Total Glutathione (GSH + GSSG) Assay

1. Add 20 μL GSH standard or sample supernatant to each well. Two or three replicate wells are recommended.

2. Add 120 μL Buffer Solution, 20 μL Substrate Solution, and 20 μL Coenzyme Solution to each well and mix gently.

3. Incubate at 37°C for 5 min, then add 20 μL Enzyme Solution to each well to initiate the reaction.

4. Immediately measure absorbance at 412 nm and continue reading once every minute for a total of 5 min. Use data from the linear reaction range for both standards and samples.

GSSG Assay

1. Mix 100 μL GSSG standard or sample supernatant with 5 μL Masking Solution and incubate at room temperature for 60 min to block GSH in the sample.

2. Add 5 μL Quenching Solution, mix thoroughly, and incubate at room temperature for 10 min.

3. Transfer 20 μL of the treated standard or sample and perform Steps 2-4 of the Total Glutathione Assay.

Calculation

Determine total glutathione and GSSG concentrations from the corresponding standard curves. Reduced GSH is calculated as: GSH = total glutathione - 2 × GSSG. Multiply by the sample dilution factor and normalize to sample volume, cell number, tissue weight, or protein content as required.