I differentiated THP-1 cells with PMA into macrophages during 72 hours. Then, I washed with PBS and detached them from the flask. After I got the THP-1 derived macrophages, I cocultured them with CD4+ T cells. Then, I realized that because of PMA, CD4 on T cells was downregulated . Although I washed THP-1 derived macrophages several times, I could not wash away PMA from the culture. So, how can I get rid of PMA from the culture, do you have any idea about it?
Do you do the enhanced PMA treatment of Deignault where you leave them for a few days without PMA? That may help together with more washes perhaps in medium, not PBS as PMA is not soluble in aqueous solutions but should bind to albumin in culture medium.
Actually, after I collected the cells, I washed them with RPMI 1640 two times. Now, I have checked that RPMI 1640 does not contain albumin. Do you think that if I incubate the cells with albumin, will it be beneficial to get rid of PMA?
The RPMI has no albumin but the serum in the medium will. That should help get as much PMA out as possible
It is my past recollection that it was considered virtually impossible to remove PMA from a culture. Some people used PHA instead since that could be washed out. You might want to contact Peter Blumberg at the National Cancer Inst. as he is a real expert in PKC activtion
It is indeed not worth trying to remove PMA. Depending on your needs you might try stimulating with phorbol dibutyrate (PDB) instead. It performs well, is reasonably water soluble and rinses out fairly well.
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