Posted 17 August 2011 - 09:12 AM
I am trying to differentiate THP-1 into macrophages using PMA.
Coul you tell me how can I detache macrophages?
Many thanks in advance!!
Posted 18 August 2011 - 12:02 PM
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.
Posted 25 August 2011 - 07:54 AM
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?
Posted 03 October 2011 - 12:45 AM