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[lamaksha77]

I  was previously working with primary monocytes, which I activated using interferon gamma treatment (and then infect with an intracellular bacterium). Now, I am switching to the pro-monocytic U937 cell line for some follow up studies, so I am trying to figure out a protocol to differentiate the U937 cells into activated macrophages before I infect them.

I tried using interferon gamma (20ng/ml) treatment for 48hs, but although the cells did seem to change shape (around 50% were spindly, or non-rounded), they were not adherent and mostly got washed away when I changed media prior to infection.

On the other hand, when I add PMA (10ug/ml) for 2 days, the cells adhere well, but they are fully rounded and no morphological change can be seen, so I am worried as to whether these adherent cells are truly activated…

Does anyone here have some experience with plugging in the interferon gamma into this differentiation protocol? Also, does my basic PMA treatment (10ug/ml for 48h) seem ok, and is it expected to see some change in cell morphology upon monocyte differentiation?

Thanks for any advice!