ICT’s Pyroptosis/Caspase-1 Assay Kits utilize our popular FLICA® technology to detect caspase-1 activation. These kits contains the caspase-1 inhibitor reagents, which have the preferred binding sequence for caspase-1, Tyr-Val-Ala-Asp (YVAD). These kits also contain Nigericin, a potent microbial toxin derived from Streptomyces hygroscopicus, that acts as a potassium ionophore, capable of inducing a net decrease in intracellular levels of potassium which is crucial for oligomerization of the NLRP3 inflammasome and activation of caspase-1.
Our Pyroptosis/Caspase-1 Assay, Green can be used 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.
Our Pyroptosis/Caspase-1 Assay, Far Red can be used 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.
Nigericin can be utilized as a positive control in pyroptosis experiments. As mentioned above, Nigericin is a potent microbial toxin derived from Streptomyces hygroscopicus. It acts as a potassium ionophore, inducing a net decrease in intracellular levels of potassium, which is crucial for oligomerization of the NLRP3 inflammasome and activation of caspase-1. It requires signaling through pannexin-1 to induce caspase-1 activation and IL-1ß processing and release. It has been shown to generate a robust caspase-1 activation response in various cell lines, including Jurkat and THP-I cells.
To learn more about pyroptosis and ICT's Pyroptosis kits, read our educational piece "Introduction to Pyroptosis" here.
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ICT products are intended for research only, not for use in diagnostic procedures.