We are trying to reproduce a model of MLRP3 activation. Based on literature protocols, we prime peritoneal murine C57bl/6j macrophages with 1 ug/ml LPS in DMEM or RPMI1640 for 3 hr., then incubate with test compounds and finally activate NLRP3 with 5 mM ATP for 30-45 min. IL-1beta is then determined by ELISA.
The thing is, we observe only insignificant up-regulation of IL-1beta (10-15% above control cells). May be we are missing something?
I do not know whether your test compound is supposed to inhibit or activate the NLRP3 inflammasomes but if your test compound can inhibit any step in priming of IL-1beta then I think there would not be sufficient IL-1beta for secretion following inflammasomes activation. But if your compound is supposed to increase priming then it would be a different scenario. I think the dose of both LPS and ATP is fine, however, the duration of priming maybe a bit short. So, try to increase the LPS treatment duration in a time-dependent fashion and pick the suitable point. Also, I don't understand why would you consider a 15% increase as insignificant. But, anyway I think increasing the priming time would generate sufficient pro-IL-1beta for further maturation via ATP-dependent inflammasomes activation at a later phase. So, it might work. Also, it is possible that either LPS or ATP or your compounds have gone bad and lost its potency.
Hope it helps.
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