1. Standard Curve: Add 10 µL of the 20 µg/µL standard GSH stock to 990 µL of Assay Buffer to generate 0.2 µg/µL working standard solution. Add 0, 2, 4, 6, 8, 10 µL to a 96-well plate to generate 0, 0.4, 0.8, 1.2, 1.6, 2.0 µg/well GSH. Bring the volume to 90 µL with Assay Buffer. Note: If the concentration of your assay samples is lower than the above standard range, the standard can be further diluted 10-fold to generate 0, 40, 80, 120, 160, 200 ng/well GSH by following the same procedure.
2. Preparation of Samples for Assays: Add 20 µL of ice cold 3N KOH to 40 µL of PCA preserved samples (as prepared in Section IV) to precipitate PCA and neutralize the samples (pH should be 5-10). Keep on ice for 5 min then spin 2 min at 13,000 G at 4 °C. Transfer 10 µL of the neutralized samples to a 96-well plate. You may choose to add several dilutions (e.g., 1-10 µL) of your samples to ensure the readings are within the standard curve range. A. To Detect GSH: Bring the sample volume to 90 µL with Assay Buffer. B. To Detect Total Glutathione: Bring the sample well to 80 µL with Assay Buffer. Do a background control without sample. Add 10 µL of Reducing Agent Mix to the wells, mix well, incubate at room temperature for 10 min to convert GSSG to GSH. C. To Detect GSSG: Bring the sample well volume to 70 µL with Assay Buffer. Do a background control without sample. Add 10 µL of GSH Quencher, mix well, and incubate at room temperature for 10 min to quench GSH. Then add 10 µL of Reducing Agent Mix to destroy the excess GSH Quencher and convert GSSG to GSH.
3. Assay: Add 10 µL of OPA Probe into the standard and sample wells, mix well, incubate at room temperature for 40 min. Read samples and standards on a fluorescence plate reader equipped with Ex/Em = 340/420 nm. We suggest adjusting the plate reader settings so that the background reading without glutathione is at about 50-150 RFU.