Intracellular GSH Assay Kit

1. Prepare samples and controls., 2. Dilute 10X Binding Buffer 1:10 WITH DiH2O., 3. Wash cells twice in ice-cold culture medium or PBS and resuspend in ice-cold 1X Binding Buffer at 5 x 105 – 1 x 106 cells/mL., 4. Reconstitute Bovine Annexin V-Fluorescein in 1 mL Annexin Reconstitution Buffer, 1X., 5. Dilute reconstituted Annexin V-Fluorescein 1:5 in PBS., 6. Add diluted Annexin V-Fluorescein to each sample at ~1:10., 7. Dilute PI 1:10 in PBS., 8. Add diluted PI to each sample at ~1:20., 9. Incubate 15 minutes on ice and protected from light., 10. Add 400 µL 1X Binding Buffer to each sample., 11. Analyze with a flow cytometer. Annexin V-Fluorescein excites at 494 nm and emits at 521 nm. PI excites at 536 nm and emits at 617 nm.