Pyroptosis/Caspase-1 Assay - Green Fluorescent

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.