Dear all,
I am currently doing human macrophage differentiation and polarization (to M1 and M2) from CD14+ monocytes. I isolate CD14+ monocytes from human PBMCs with miltenyi beads, then I incubate with 50ng/ml hGM-CSF (R&D Systems) for M1-macrophages and hM-CSF (R&D Systems) for M2-macrophages. After 3 days, I refresh media+factors, and on day 6, I activate M1-macrophages with LPS (50ng/ml; R&D Systems) and IFNg (50ng/ml; R&D Systems) and M2-macrophages with IL-4 and IL-13 (20ng/ml; R&D Systems). On day 7 I use them for different experiments.
The problem is that sometimes I obtain really nice polarized macrophages (I already characterized them), but around 50% of the times I have the problem that monocytes incubated with GM-CSF or M-CSF don't differentiate to macrophages, and I don't know which is the problem --> so I mean that sometimes work well for GM-CSF but no for M-CSF or the other way around. I never change conditions or any step of the protocol, and sometimes work well, sometimes it doesn't work well. Have you seen anything similar? I thought maybe the donor was the variable, but it happened also to me with the same donor (the first time it worked perfectly for both M1 and M2, and the second time with the same donor I got nice M1-macrophages, but I didn't obtain M2-macrophages, they never attached). I also thought about the GM-CSF or M-CSF batch, but with the same batch, sometimes works, sometimes doesn't work. So the problem is that I don't get what I do or what is different from one time to another.
Any suggestion will be welcome, and many thanks in advance!
Jana
Hi Divya, thanks for your answer. Yes, and actually this is the only thing I think it can be my problem, but still not sure. So in the beginning, I check 2-3 times the purity by flow cytometry, and it was between 75 and 85%, so not super great (I was expecting a bit more). My theory is that as less purity I have, as more problems I have to differentiate them. I say that because I realize that when I obtain more "CD14 cells", then normally it goes worst. When I have less number (so less percentage), normally it goes better. Maybe other cells uptake the GM-CSF or M-CSF, then monocytes can't differentiate well? Tomorrow I will actually run a single round of monocyte isolation versus a double round of monocyte isolation (so passing cells 2 times through the column), to see if I increase the purity, it goes better. If you want, I can tell you the result.
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