Hi,
I'm transducing Ly1 and L363 cell lines using our standard protocol for retroviral transduction. The cells are successfully transduced as evidenced by GFP expression. However, after 4-5 days they start dying off and look really stressed. I'm suspecting polybrene since we've got a new batch. The cells look really weird, irregular and start forming clumps which they don't normally do in standard cell culture. I've tried using the same polybrene concentration (8ug/ml) in standard culture medium without the virus to check toxicity and it appears that it is decreased. Which concentrations do you normally use? Should I make a polybrene concentration curve to find the minimal nontoxic condition?
its not the transfected protein that is toxic to the cells though? You have transduced with that one before?
I suspect that your problem is the polybrene, since your RV is only GFP+, and would try to titrate the polybrene concentration. But I did have a bizarre experience with transducing two different cell lines with RV when I was studying the immunosuppressive mouse retrovirus LP-BM5 decades ago. The plan was to transduce the virus into H2b (C57BL/6) vs H2d (BALB/c) fibroblasts to a) determine if the strain-specificity of the virus (B6 are sensitive, BALB are resistant) was due to differential infectibility or virus production, and b) generate target cell lines for CTL assays.
I got cell lines from ATCC and transduced them with 8 ug/ml polybrene, and the results were strikingly different. The transduced BALB fibroblasts transduced nicely and produced infectious virus and maintained their viability, growth pattern and morphology. The transduced B6 morphology was drastically altered, their growth rate was much reduced and their viability was low. The cells produced infectious virus, but never grew well after that. I do not think this is your problem, but I'm not familiar with Ly1 cells. I'm guessing your L363 are derived from L cells?
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