Hi,
I'm trying culturing monocyte, isolated from PBMC's to differentiate them in macrophages. I obtained a buffy coat from a healthy donor, I freezed most of cells in freezing solution (90%FBS+10%DMSO) and I put 10 millions cells in a 6 well plate. The day after I threw away cells in suspension and I added 2ml of RPMI-1640 10%FBS+1%L-Glut+1%P/S with 20ng/ml of M-CSF in the well. Six day after, adherent cells looked like macrophages (picture in attachment) and I added citokynes for differentiation for 24h. I repeated the experiment using PBMC's I freezed previously. After thawed cells, I performed a positive separation for CD14+ with beads. I plated cells in X-VIVO-15 2%FBS+L-Glut1%+1%P/S. Today, 5 days after the seed, cells are round and in suspension, and their viability with trypan blue is about 30%. What was the problem? I read monocytes freezed have a good yeld, and it's better using x-vivo-15 medium for macrophages differentiation. Maybe the percentage of serum it's too low?
Thanks
Dear Federico. It is possible that the problem is the form used to freeze PBMC Cells. In our case, we used RPMI medium plus 20% AB human serum and 10% DMSO Best regards, Gustavo.
Dear Federico. Thank you for your interesting question. Are you seeding 10 million cells in total per plate or per well? One well should be around 9.6 cm^2 (please correct me if I am wrong here). The number of cells should be fine if you go with 10^6 cells per square cm flask/plate. IMO the number of cells you were using might be just to low. When I was using PBMCs, I was doing FC just before and was checking for the monocyte frequency (just by SSC/FSC) in the suspension; if the monocyte frequency was below 25%, was was plating 1.5x10^6 cells per square cm, if it was above 25%, I was using 10^6 cells.
For adherence, cells were incubated on the plate for one our in cell free PBS, then washed with PBS and PBS finally exchanged with medium + heat inactivated serum.
Worked fine for me. Hope this information helps you. Found some old image of my cells after 5d, however, I do not know why the image is in orange.
Cheers!
Johannes Zeller Thanks for your answer.
Yes, I seeded 10 million cells per well. Next time I'll use a medium FBS free to increase the attachment rate. Can I ask you the percentage of serum you used to culture your cells? Thanks
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