I'm in need of a bit of help - I've been culturing them in RPMI-1640 with 10% FBS and attempting to induce differentiation with 10nM PMA. I generally seed them at a density of 100,000 cells per well in poly-L-lysine-coated 24 well plates. This protocol used to work, but after going on vacation, the cells will no longer cooperate. I've tried changing the cell density and ordering a new stock of PMA and poly-L-lysine. I thought the likely cause was infection, but I've sterilized the incubator and tried multiple U937 stocks from different batches. I'm wondering what else could cause this - maybe endotoxin-contaminated FBS? Anyhow, there's a deadline coming up for a paper, and I'd really appreciate any advice. Thanks in advance!
Hi Michael.
I used to do U937 differentiation to macrophages by adding PMA at 160 nM final concentration, and then let them sit for 48 hrs under culture conditions, when they change their phenotype to adherent cells more like macrophages.
I reconstituted PMA using DMSO or 100% ethanol and you should always remember that it is light sensitive, so I never used one aliquot twice always avoiding freeze-thaw cycles and light exposure.
Briefly, for a six well plate I differentiated 1 x 10^6 cells in 2 mL of RPMI supplemented with 10% FBS and PMA (160nM), so I will get around 50-60 differentiation after 48 hrs. TO NOTE: Before I transfer cells to the plate, I always swirl manually the mixture cells:media:PMA around 1 min and then I transferred them to the plate.
Hope these tips help you in differentiating U937s.
Best, HEnry
So PMA is that sensitive? I tried buying a new stock altogether, but that didn't resolve the problem. I wound up moving onto a new cell line, but thank you for the response
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