I use 10% human serum, 1% penicillin streptomycin RPMI 1640. Isolate PBMC from buffy coat and culture with or without stimuli. T cells are 60-70% after two days and 1-7% B cells, no monocytes by size (forward and side scatter). I started to work with human samples recently but is it normal? I feel B cells are too low and there should be monocytes population. Any suggestions appreciated.
Thank you Laila-Aicha, I am doing flow for now but I will start activation and differentiation too so I need them to be alive and healthy. Good to know that T cells dominate in general. I had soluble CD40L in few cultures but they were not very alive either. Does IL-2 help B cells too? I used to add IL-2 for T cells but I am very new to B cells. I ordered the non-stick plate so see monocytes. Thank you so the references, these are very helpful!
B cells will die in culture without addition of cytokines (eg. IL-4, IL-21, IL-6, BAFF) and/or stimulation through the B cell receptor or other TLR ligands. Many of the combinations that improve B cell survival in vitro also induce activation/differentiation of these cells into antibody secreting cells. Depending on your question this may or may not be desirable.
Fixed Staph Aureus (also called SAC in literature) or (Fab)2 anti-IgM + cytokines will help you get more B cells. Pokeweed mitogen is another agent.
All the cytokines mentioned above will help. For human B cells IL-10 is also a good cytokine to include. Keep in mind that in PBL only 5-15% are B cells. Best way will be to enrich for B cells first.
Thank you Andrew and Subbarao, sorry for the late response.
I do want to study activation/differentiation as well but that will change the surface marker expression. If they do not survive without stimulation, I need to do it with stimulus and activating and differentiation conditions.
Enriching for B cells is a good idea but if there are other cells, it could help B cell survival as well? I heard that B cells in PBMC survive but purified B cells don't.
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