I'm trying to induce inflammasome activation in THP1. I added 100ng/ml PMA to induce differentiation of THP1 for 24 hours, then change medium without PMA and rest for 24 hours. I added 1ug/ml LPS for 4 hours, followed by 10nM nigericin for 1 hour. I used WB to detect NLRP3 expression but found no diference between ctrl and LPS+nigericin group. Does anyone have similar problem?
Hi! maybe your concentration of PMA is too high? even with the rest period I have noticed increased baseline NLRP3 expression in THP-1 cells differentiated in 100nM PMA (162ng/ml) compared to 25nM (15.4ng/ml).
Giambelluca et al recently published on this:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9817155/
you can use 2.5 ng/ml of PMA to differentiate THP-1 cells for 48 hours, followed by 3 hours of starvation in serum-free media. For LPS stimulation I use 50ng/ml LPS and it works well for me.
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