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.
Product Specific References
|37123216||Li, H., et al. 2023. Heme oxygenase‑1 inhibits renal tubular epithelial cell pyroptosis by regulating mitochondrial function through PINK1. Experimental and Therapeutic Medicine, 213.|
|37093343||Liang, M.Q., et al. 2023. LncRNA SNHG3 Promotes Sevoflurane-Induced Neuronal Injury by Activating NLRP3 via NEK7. Neurochemical research.|
|37182453||Yan, C., et al. 2023. Endoplasmic reticulum stress promotes caspase-1-dependent acinar cell pyroptosis through the PERK pathway to aggravate acute pancreatitis. International immunopharmacology, 110293.|
|37356296||Mikolajczyk-Martinez, A., et al. 2023. Unraveling the Role of Type 1 Fimbriae in Salmonella Pathogenesis: Insights from a Comparative Analysis of Salmonella Enteritidis and Salmonella Gallinarum. Poultry Science, 102833.|
|37602503||Zou, H., et al. 2023. C/EBPβ isoform-specific regulation of podocyte pyroptosis in lupus nephritis-induced renal injury. The Journal of pathology.|
|37556724||Torraca, V., et al. 2023. Shigella serotypes associated with carriage in humans establish persistent infection in zebrafish. The Journal of infectious diseases.|
|35151118||Zhang, C., et al. 2022. Targeting NLRP3 Signaling by a Novel-designed Sulfonylurea Compound for Inhibition of Microglial Inflammation. Bioorganic & Medicinal Chemistry, 116645.|
|35180562||Zeng, M., et al. 2022. LncRNA ROR promotes NLRP3-mediated cardiomyocyte pyroptosis by upregulating FOXP1 via interactions with PTBP1. Cytokine, 155812.|
|35721108||Wu, X., et al. 2022. Emodin Ameliorates Acute Pancreatitis-Associated Lung Injury Through Inhibiting the Alveolar Macrophages Pyroptosis. Frontiers in pharmacology, 873053.|
|35835255||Bertoni, A., et al. 2022. Spontaneous NLRP3 inflammasome-driven IL1-β secretion is induced in severe COVID-19 patients and responds to anakinra treatment. The Journal of allergy and clinical immunology.|
|35761192||Lan, J., et al. 2022. WTAP-mediated N6-methyladenosine modification of NLRP3 mRNA in kidney injury of diabetic nephropathy. Cellular & molecular biology letters, 51.|
|36129987||Duhalde Vega, M., et al. 2022. PD-1/PD-L1 blockade abrogates a dysfunctional innate-adaptive immune axis in critical β-coronavirus disease. Science advances, eabn6545.|
|36155068||Shu, L., et al. 2022. PHLDA1 promotes sevoflurane-induced pyroptosis of neuronal cells in developing rats through TRAF6-mediated activation of Rac1. Neurotoxicology, 140-151.|
|36357533||Clark, K.M., et al. 2022. Chemical inhibition of DPP9 sensitizes the CARD8 inflammasome in HIV-1-infected cells. Nature chemical biology.|
|36429008||Liu, Y., et al. 2022. S100A8-Mediated NLRP3 Inflammasome-Dependent Pyroptosis in Macrophages Facilitates Liver Fibrosis Progression. Cells, 3579.|
|33667846||Lu, R., et al. 2021. Design, synthesis and evaluation of 3-amide-5-aryl benzoic acid derivatives as novel P2Y14R antagonists with potential high efficiency against acute gouty arthritis. European Journal of Medicinal Chemistry, 113313.|
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.