Necrosis vs Apoptosis Assay Kit

1. Prepare samples and controls, 2. Dilute Apoptosis Wash Buffer 1:10 with diH2O., 3. Reconstitute FAM-VAD-FMK with 50 µL DMSO., 4. Dilute FLICA 1:5 by adding 200 µL PBS., 5. Add diluted FLICA to each sample at 1:30 (e.g., add 10 µL of diluted FLICA to 290 µL of sample)., 6. Incubate approximately 1 hour., 7. Remove media, then wash cells: add 1X Apoptosis Wash Buffer and spin cells (twice)., 8. Resuspend samples in 400 µL 1X Apoptosis Wash Buffer, 9. Reconstitute 7-AAD with 260 µL DMSO, 10. Add 7-AAD to each sample at 1:200 (e.g., add 2 µL to 400 µL of sample), 11. Analyze with a fluorescence microscope or flow cytometer. FAM-VAD-FMK excites at 492 nm and emits at 529 nm. 7-AAD excites at 546 nm and emits at 647 nm.