I've read a number of protocols on this and several people's comments, but try as we might our lab is struggling to make this a success; whether we are culturing to differentiate macrophages (conditioned L929 media) or dendritic cells (GM-CSF), we're left with virtually zero viable cells at the end. I suspect that the hematopoetic progenitors are not surviving the process.
After I collect the bone marrow, I wash them once with complete DMEM and then resuspend in 90% FBS + 10% DMSO. Aliquot the cells (10^7/tube) into cryotubes and then into rate-controlled freezing container overnight and transferred to liquid N2 the next day. For recovery, we put the frozen tube into 37 deg waterbath until *almost* thawed, transfer the cell slurry into pre-warmed complete media (~20-30 mL), centrifuge to remove DMSO, and resuspend in differentiation media.
The only things I can think of that might still be affecting our results is 1) the concentration of cells/freezing media may be too high or too low, 2) thawing is too quick (although I thought that was the point), 3) handling is too rough; the cells need a rest period before differentiation.
But truthfully, I have no idea. By all accounts I've heard, we should at least be getting SOMETHING. When we prepare macrophages or DCs from fresh bone marrow, all is well and our yield is excellent.
Does anybody have any thoughts? What the heck are we missing here?
Hello
See these links
Good luck
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3003694/
https://www.ncbi.nlm.nih.gov/pubmed/12191518
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0015263
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