This in vitro assay employs the far-red fluorescent inhibitor probe 660-YVAD-FMK to label active caspase-1 enzyme in living cells. Analyze samples using fluorescence microscopy, or flow cytometry.
Samples:
Cell culture
Application Notes:
Application Notes: 1. Prepare samples and controls, 2. Dilute 10X Binding Buffer 1:10 with diH2O., 3. Wash cells in ice-cold culture medium or PBS and resuspend in ice-cold 1X Binding Buffer., 4. Dilute Annexin V-FITC 1:10 in PBS., 5. Dilute Propidium Iodide (PI) 1:2 in PBS., 6. Add diluted Annexin V-FITC to each sample at a ratio of 1:20 (5 µL per 100 µL aliquot of cultured cells)., 7. Add diluted PI to each sample at a ratio of 1:20 (5 µL per 100 µL aliquot of cultured cells)., 8. Incubate ~10 minutes, protected from light., 9. Dilute samples by adding 250 µL 1X Binding Buffer to each., 10. Analyze with a flow cytometer. Annexin V-FITC optimally excites at 494 nm and emits at 519 nm (typically read in FL1). PI optimally excites at 536 nm and has a peak emission at 617 nm (typically read in FL3)
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