Method of Analysis:Flow Cytometry, fluorescence microscope, fluorescence plate reader
Intended Use:Pyroptosis/Caspase-1 BioAssay™ Kit utilizes our popular FLICA technology to detect caspase-1 activation. Using this BioAssay™ kit, researchers can easily assess pyroptotic cells and utilize nigericin as a positive control. Analyze the green fluorescent signal using fluorescence microscopy, a fluorescent plate reader, or by flow cytometry.
Test Principle:Inflammatory caspases, such as caspase-1, play a critical role in the mechanism to trigger the lytic programmed cell death known as pyroptosis. Pyroptosis/Caspase-1 BioAssay™, Green can be used by researchers to assess caspase-1 activity in pyroptotic cells and utilize nigericin as a positive control.
The FLICA reagent FAM-YVAD-FMK enters each cell and irreversibly binds to activated caspase-1. Because the FAM-YVAD-FMK FLICA reagent becomes covalently coupled to the active enzyme, it is retained within the cell, while any unbound FAM-YVAD-FMK reagent diffuses out of the cell and is washed away. The remaining green 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 fluorescent plate reader, fluorescence microscopy, or flow cytometry. Cells labeled with the FLICA reagent may be read immediately or preserved for 24 hours using the fixative. Unfixed samples may also be counter-stained with Propidium Iodide, 7-AAD, or Hoechst (included in the kit).
Nigericin induces a net decrease in intracellular levels of potassium, crucial for activation of caspase-1. In pyroptosis experiments, nigericin can be used as a positive control to generate a robust caspase-1 activation response in a variety of cell lines.
Storage and Stability:Store powder at 4°C liquid at -20°C. Store other components at 4°C. Stable for 6 months For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap.
