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.