I am referring to conventional dendritic cells from the mouse lymph node, not bone marrow derived dendritic cells. I don't know what to put in the culture medium. GM-CSF will result in DC proliferation and derivation of monocyte derived DCs which will confound my cell counts in the survival counts. But how do I keep them in culture for a reasonable period of time to allow assessment of longevity?
Addition of Flt3L may be a solution for improving the longivity. A combination of Flt3L with a very low dose of GMCSF may be better. [there are some studies on the role of Flt3L and GMCSF concentration on BM DCs and that may be applicabale for your DCs- PMID: 21422244 ]
Hello Dunja,
If I am able to understand correctly, you want to culture cells obtained from mouse lymph nodes and determine their vaibility (longevity in your terms) without getting them transformed. I would suggest to keep them into media (e.g. RPMI), in a multi well plate and do viability check every other day in triplicates at least.
I would say that viability will start reducing after 3rd day and cells would be live max. till 1 week. I am just suggesting this from my experience with mouse lymph node and PBMC derived DCs in separate experiments.
Hope it helps.
Hi Sreekumar, thanks for the tips. I will try this with Flt3L and low dose GMCSF.
Varun, yes but I will be sorting immature splenic DCs specifically and 70% of them usually die within 24h of culture.
I am not sure i understand your procedure; it seems that you are sorting the DC subsets and keep them in culture and look for the viable cells.
One of the major reason for cell death may be the sorting procedure. Few tips like sorting DCs at a low PSI (100µ nozzle /20PSI pressure), Sorting cells in 1X PBS+ 1mM EDTA+ 15 mM HEPES instead of sheath fluid. use of pretreated collection tube- coating the tubes overnight with 2%BSA etc may really improve the cell viability.
Yes we use a 100ul nozzle generally. But I was going to MACS sort them initially for a test run. The sorting is not usually the issue, its just that the DCs will die if they do not mature with antigen (http://www.ncbi.nlm.nih.gov/pubmed/9016880, http://www.ncbi.nlm.nih.gov/pubmed/9841930 ).
I just want to compare general viability of immature DCs between WT and knockout mice.
http://www.ncbi.nlm.nih.gov/pubmed/25081090
Maintaining dendritic cell viability in culture.
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