I wish to evaluate some membrane antigens on macrophages differentiated from THP-1 cells. My differentiation protocol consist of 50ng/ml PMA for 72hs and then, after washing out non-adherent cells, one day with RPMI medium.
Another possible method is to use a solution of EDTA without Trypsin and incubate for 5 minutes. This will dislodge most of the cells, and whatever remains can then be scrapped as mentioned in the post above. This may help preserve some of the surface markers you may be flowing for.
Hi Tomas: Both Shann and Ryan´ answers are complementary. Ryan'answer is better because surface markers are an important outcome in this procedure.
we use PBS with 5mM EDTA for 10 mins for detaching the differentiated THP1
Hi everyone, I know the topic is relatively old, but i'm working on THP-1-derived macrophages M1 and M2, essentially to assess flow cytometry, but the issu is the harvest of M1 macrophages. Cause when I detach cells, almost all my M1 macrophages died. I tried all different types of techniques (EDTA 5mM, 5min, 30 min (4 degrees), with/without cell scraper, cold / warm PBS). No way, all my M1 died immediately after being detached.
Any suggestions? Cause markers are relatively altered in death cells, mostly in M1 cause they're inflammation specifics, and change quasi instantly, and with 5M cells at the beginning, around 90% died at the end.
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