How can I measure NLRP3 inflammasoem activation? In most of the papers, I found protein and mRNA expression levels of NLRP3 and caspase-1. Since NLRP3, Asc and procaspase-1 make an assembly and it is known as inflammasome activation; is there by any means I can measure this assembly formation?? or will the molecular size of NLRP3 be affected in general SDS/PAGE if there is inflammasome activation?
Protein and mRNA levels are not appropriate to measure NLRP3 activation. You should use the formation of ASC or NLRP3 specks, cleavage of caspase-1 or IL1b by Western blot, or release of IL1b by ELISA as indicators. Specificity can be achieved by specific inhibitors. I've used these methods and provided detailed protocols in the following papers:
Article K+ regulates Ca2+ to drive inflammasome signaling: Dynamic v...
Article The oxindole Syk inhibitor OXSI-2 blocks nigericin-induced i...
The titles are in my post, and the PDFs can be accessed from my profile.
Hi Jordan, I went through your papers. I still want to understand few concepts. Why do we measure asc oligomer formation? I know that asc makes oligomers with both NLRP3 and NLRP1. If I only want to show the assembly of procaspase-1, NLRP3 and asc, is there any specific assay for that???
There are colorimetric kits that can detect Caspase-1 activity. They work on cell lysates.
Also IL18 secretion. It is very cell dependent
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