I activated my BV2 microglia cells with LPS (500 ng/ml) followed by 45 minutes incubation with 5 mM ATP. Although the IL-1B mRNA was 2000-fold increased regarding the untreated cells, I didn't detect any expression of this protein in the cell supernatants using a mouse IL-1B ELISA kit (standard curves were perfect). Anyone has any idea of what is happening?
Hi Maria,
In our lab we don't use BV2 cells because we have seen that they're not able to activate the NLRP3 inflammasome. Although I don't have papers to support this (just experiments from our lab), if you look for papers using BV2 in NLRP3 activation you will see few papers and always around 50 pg/ml release of IL-1b, which is quite low in comparison to different cell types that release between 500 to 5000 pg/ml.
If your context is microglia and IL-1b release the primary mixed glia culture is a good model, but if you only need an immortal cell line you could use human THP-1 or mouse immortalised BMDMs.
Finally, if you keep trying with BV2 cells you could check intracellular levels of IL-1b, NLRP3 and ASC by western blot, just to be sure you have all the components for IL-1b release.
Victor S Tapia, thank you so much for your answer. As you suggest I'll check by western the intracellular levels of these proteins, and I'll try a different cell line
Thanks again!
I am on same boat, i can see some papers on this. It it does not worked for me, any suggestion. Your advise will help me designing future experiments on this. Thanks
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