ICT’s Pyroptosis/Caspase-1 Assay, Far Red utilizes our popular FLICA technology to measure caspase-1 activation. Using this kit, researchers can easily detect pyroptotic cells in their whole cell samples in vitro. This complete kit includes Nigericin for the simple and reliable generation of a positive control. Analyze the fluorescence signal using flow cytometry or fluorescence microscopy.
Inflammatory caspases, such as caspase-1, play a critical role in the mechanism to trigger the lytic programmed cell death known as pyroptosis. ICT’s Pyroptosis/Caspase-1 Assay, Far Red can be used by researchers to assess caspase-1 activity in pyroptotic cells. This kit utilizes the far red, cell permeant FLICA reagent, 660-YVAD-FMK, for the in vitro detection of caspase-1 in whole living cells. 660-YVAD-FMK enters each cell and irreversibly binds to activated caspase-1. Because 660-YVAD-FMK becomes covalently coupled to the active enzyme, it is retained within the cell, while any unbound reagent diffuses out of the cell and is washed away. The remaining far red fluorescent signal is a direct measure of the active caspase-1 enzyme activity present in the cell at the time the reagent was added. Cells that contain the bound FLICA can be analyzed by a fluorescence microscope or flow cytometer. Cells labeled with the FLICA reagent may be read immediately, or preserved for up to 16 hours using Fixative (included in the kit). Unfixed samples may also be counter-stained with Hoechst 33342 (included in the kit) to label cell nuclei. This complete kit also includes Nigericin as a convenient option for generating a positive control. Nigericin induces a net decrease in intracellular levels of potassium, crucial for activation of caspase-1. In pyroptosis experiments, it has been shown to generate robust caspase-1 activation in a variety of cell types.
660 nm / 690 nm
Flow cytometry, Fluorescence microscope
Domestic: Overnight Delivery; International: Priority Shipping
- Prepare samples and controls.
- Dilute 10X Cellular Assay Buffer 1:10 with diH2O.
- 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 sample).
- Incubate approximately 1 hour.
- Remove media and wash cells 3 times: add 1X Cellular Wash Buffer and spin cells.
- Resuspend cell pellet in 1X Cellular Wash Buffer.
- If desired, label with additional stains, such as Hoechst 33342, DAPI, or an antibody.
- If desired, fix cells.
- Analyze with a fluorescence microscope or flow cytometer. FLICA 660 is excited at 660 nm and emits at 680-690 nm.
Mao, x;Wu, W;Nan, Y;Sun, W;Wang, Y. SMAD2 inhibites pyroptosis of fibroblast-like synoviocytes and secretion of inflammatory factors via TGF-β pathway in rheumatoid arthritis. Research Square. 2023 January; doi: 10.21203/rs.3.rs-2471290/v1. PDF