Dear colleagues, currently I am doing an inflammasome-related experiment using cells. I have a substance as a candidate for anti-inflammasome. However, I am a little bit confused about which step I should administrate my candidate regarding the inflammasome molecular pathway. Is it better after primary inducer (LPS) and before secondary inducer (ATP) or simultaneously administrate with the secondary inducer (ATP)? I beg your suggestion and further consideration. Thank you.
Hello
I think it depends on which step your compound acts. Maybe it can inhibit the priming step, so you should treat your cells with it, 1 or 2 hours before LPS priming. If your compound acts on ATP signaling, I think you can add your inhibitor along the last 1 or 2 hours of LPS treatment to be sure that it will inhibit NLRP3 activation. I think a pre-treatment is more sure, as ATP needs only few minutes to activate NLRP3 (if you work on macrophages).
Best.
I agree with the previous comments. I would like to add that if the potency of the "substance" as an anti-NLRP3 inflammasome activation is not known, optimization of treatment may be needed- treat before "priming" and after the "priming" assuming that the "substance" does not alter the levels of NLRP3, ASC, and pro-caspase-1 in your cells.
I third the prior statements about optimizing empirically.
To answer your question based on my experience and with the inhibitors I worked with, in my ATP-based experiments I did my pretreatments in the last 15-20 minutes of my LPS priming prior to ATP stimulation. You can see ATP's action on cells within a few seconds, the assembly of the inflammasome occuring only a few minutes later.
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