THP-1 Differentiation - (Aug/17/2011 )
I am trying to differentiate THP-1 into macrophages using PMA.
Coul you tell me how can I detache macrophages?
Many thanks in advance!!
We wash them twice in Ca++-free PBS, put in just enough more PBS to cover the cells, and scrape them with a plastic cell scraper. It's much less damaging (though not entirely non-damaging) to these cells than trypsin, as well as faster and giving a better cell yield.
Another tip, we usually expose our macrophages to PMA overnight, then wash away the PMA and non-adherent cells. Then we mature the adherent cells in fresh THP-1 growth media for 7 days prior to using the macrophages (refresh media every 2-3 days). That increases cell yield (they do divide, if much slower than the original monocytes), and stabilizes the results better. They're going to keep differentiating for at least 5 days, so your results can vary with tiny changes in timing if you don't give them time to reach a stable state of fully differentiated macrophages.
Thank you for your kind response.
I incubated THP-1 cells with PMA (10, 20, 40, 80 and 160 nM) for 72 hours. After that time, I removed the supernatant and I used two solutions to detach macrophages, 1- PBS+5mM EDTA for 30', and with this condition, I get macrophages just from the 10nM of PMA. I also used trypsin during 30', and I detach a few number of macrophages.
How can I Know that at the end of 72h of incubation with PMA I have only macrophages? At microscope I didn't see differences between monocytes and macrophages...
Next time I'll try to scrape the cells..
Many thanks in advance!!!
Do you think 72h of incubation to long?
I treat my THP-1 with PMA. I prepared 50 ml medium RPMI with 10% inactivated serum, antibiotics solution and 50 ng/ml PMA. I would like to know If I can store this medium in refrigerator for a few days? MAybe I should do a fresh medium for every experiment….