Hi,
I am studying purified mice CD4 T cells, CD8 T cells and DCs etc proliferations in vitro labelled with CFSE. But total splenocyes are NOT showing proliferation. How can I observe splenocytes proliferation in culture. I am using both the ways for stimulating splenocytes either by specific antigen or anti-CD3 & anti-CD28 or ConA stimaution.
Hi,
You probably forgot to add 2-ME at the culture medium or you are gating out the proliferated populations by a single cell gate.
Yusuf
Hi,
What were your culture condition? How are your FSC SSC plots between no stimulation and stimulation?
Whether total mice splenocye proliferate? if so, please provide me related link. Thanks
Yes they do if you add aCD3 (no need for aCD28 as APCs are contain in the splenocyte mixture). Look at the experimental procedure and the Figure 5 (CFSE labeled splenocytes stimulated for 72h and observed CFSE in CD4 and CD8+ T cells) of this paper.
Hi Mohammad,
You should detect proliferation with splenocytes and even with pure CD4/ CD8 by ConA. I would suggest solving the problem first with ConA before consuming antibodies. If you would like to share a representative FCS file I can check if there is a problem with that. Culture conditions are of course essential.
Hi, for our in vivo modelling work we required splenocyte transduction followed by magnetic bead selection. We found that following selection, splenocyte viability was poor and declined rapidly despite use of fresh medium and supplementation with IL2 and BME. However, retention (and 0.45um filtration) of the spent IL2 supplemented medium which the splenocytes had been cultured in prior to selection completely resolved this limitation and typically we observed cellular doubling overnight post selection using this modified protocol.
Hey Do you think I can wait my cells proliferate during 5 days to performe a CSFE assays?
Thanks
Dear Freitas!
You must label freshly isolated cells of interest with CFSE. Then after 4-5 days you can perform CFSE Assay (FACS). Actually, after 4 or 5 days, the cells may die to an extent that you may not be able to obtain a clear data.
All the best
Stimulation with CSA/ PhA and kept in CO2 for 3 -4 day with RPMI+15% FCS. after that at least 50-65% proliferation will found.
It's very difficult to handing also during this period contamination of culture is very high changes because of primary culture.
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