PMA/ionomycine is normally use to activate immune cells before a cytokine staining, but i can't find protocols where it is use in normal cell culture for a long time.
In my case, I want to activate all immune cells (splenocytes) and treat them with or without LPS 5ng/ml during 3 days.
Can i let the cocktail PMA (50 ng/ml)/ionomycin (1 µg/ml) for 3 days in my culture ? If not, how long (maximum) can i treat cells with this cocktail ?
Hello Gwenaelle. Overstimulation of cultured cells can result in viability issues, but the association of PMA and Ionomycin has already proven to work very well inducing T cell differentiation and proliferation in long lasting cultures before a decrease in viability start to occur. This paper (http://intimm.oxfordjournals.org/content/11/4/499.full.pdf+html) mentions their experience in using the PMA/Ionomycin association in different concentrations and for different periods of time in Thymocytes of the frog Xenopus. According to their findings, T lymphocytes continued responding well to PMA/Ionomycin activation for more than 6 days. In this other paper (http://bmcgastroenterol.biomedcentral.com/articles/10.1186/1471-230X-12-172) mouse T-cells from splenocytes were cultured with PMA and Ionomycin for up to 5 days (with supplementation between days to induce Treg differentiation). So, you'll probably be fine, but I'd suggest you double check if the concentration you are choosing is Ok. In this study with splenocytes, PMA was used at 10ng/mL and Ionomycin at 250ng/mL.
Hello Gwenaelle. Overstimulation of cultured cells can result in viability issues, but the association of PMA and Ionomycin has already proven to work very well inducing T cell differentiation and proliferation in long lasting cultures before a decrease in viability start to occur. This paper (http://intimm.oxfordjournals.org/content/11/4/499.full.pdf+html) mentions their experience in using the PMA/Ionomycin association in different concentrations and for different periods of time in Thymocytes of the frog Xenopus. According to their findings, T lymphocytes continued responding well to PMA/Ionomycin activation for more than 6 days. In this other paper (http://bmcgastroenterol.biomedcentral.com/articles/10.1186/1471-230X-12-172) mouse T-cells from splenocytes were cultured with PMA and Ionomycin for up to 5 days (with supplementation between days to induce Treg differentiation). So, you'll probably be fine, but I'd suggest you double check if the concentration you are choosing is Ok. In this study with splenocytes, PMA was used at 10ng/mL and Ionomycin at 250ng/mL.
Thank you for your answer and for the bibliographic reference you give me.
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