Far-Red Fluorescent FLICA660 Caspase-1 (YVAD) Assay Kit

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).

Question: FLICA 660 optimally excites at 660 nm and has a peak emission at 685-690 nm, and Propidium Iodide excites at 615nm. Can I use the FLICA 660 and the Propidium Iodide to detect pyroptosis by Flow cytometry?, , Answer: We have done some two-color panels pairing FLICA 660 with green emission fluors, however, we have not evaluated it alongside Propidium Iodide. That said, despite emission spectra overlap between Propidium Iodide and 660-YVAD-FMK I believe it should be possible to resolve the red vs far red fluors with appropriate compensation., , I wanted to make you aware of Green Live/Dead Stain, ICT’s membrane impermeant green fluorescent vital stain for differentiating live and dead cells. This product performs similarly to Propidum Iodide, however, due to its green emission properties, it is compatible with our FLICA 660 products without the need for compensation.