I have known that RAW cell line is defective in ASC expression,and i have isolated the peritoneal macrophage from mouse. I am confused whether it is necessary to ues PMA to stimulate the cell for differentiation and then activated by LPS+ATP later?
Thanks for your suggestions!
No it is not necessary to stimulate peritoneal macrophages with PMA because peritoneal macrophages are mature macrophages. If you treat cells with PMA, it will give effect of PMA also as PMA itself activates inflammasome. So my suggestion is use only LPS+ATP.
OK,thanks for your suggestion! I have tried LPS+ATP for several times,unfortunately，the results are unexpected！
In my experiments，I want to konw whether melatonin can inhibit the NLPR3 inflammasome activated by LPS+ATP. The experiment protocol is: after 3 days later of cultured peritoneal macrophage cells, the cells were pre-treated with melatonin(100 μM/ml about 3h ), then primed with LPS (100ng/ml for 6h),and then treated with ATP (5mM/ml for 30 min). The medium was changed every time.
Is there some wrong with my protocol? I really need help!
Thanks for your suggestion!
PMA has several issues so its not a good idea to use for mature macrophages. PMA can serve a positive control in your experiment. To understand correctly the output of your experiment I think you must have positive and negative control both.
Hi Yu
You can try dose dependent effect of melatonin to see the inhibition. LPS and ATP treatment are fine. Use fresh aliqouts of LPS and ATP. Moreover use peritoneal macrophages for treatment after 2h of plastic adherence to remove non adherent cells. Use a positive control which shows inhibition in NLRP3 expression.
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