FLICA 660 Caspase-1 Assay Kit
Detect caspase-1 activity with the FLICA 660 Caspase-1 Assay Kit. This in vitro assay employs the fluorescent inhibitor probe 660-YVAD-FMK to label active caspase-1 enzyme in living cells. Analyze the fluorescent signal using fluorescence microscopy or flow cytometry.
- Prepare samples and controls.
- Dilute 10X Cellular Wash Buffer 1:10 with diH20.
- Reconstitute FLICA with 50 µL DMSO.
- Dilute FLICA 1:5 by adding 200 µL PBS.
- Add diluted FLICA to each sample at 1:30-1:60 (e.g. spike at 1:30 by adding 10 µL to 290 µL cultured cells).
- Incubate approximately 1 hour.
- Remove media and wash cells 3 times: add 1X Cellular Wash Buffer and spin cells.
- If desired, label with additional stains, such as Hoechst, DAPI, or an antibody.
- If desired, fix cells.
- Analyze with a fluorescence microscope or flow cytometer. FLICA 660 excites at 660 nm and emits at 680-690 nm.
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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.