I've tried lentivirus but didn't get enough infected cells. An article (DOI:10.1172/jci78753) describes the following method: "B6 WT splenocytes were cultured in complete medium for 24 hours. The medium was then replaced with complete medium supplemented with Polybrene (Sigma-Aldrich) at a final concentration of 5 μg/ml, and the cells were infected with lentiviral particles carrying E2F7 or E2F8, or control vector for 3 days". But about 50% cells died after 48h of incubation in complete medium supplemented with 4,5,6 μg/ml Polybrene, more cells died in higher concentrations of Polybrene. Should I change the virus -containing medium after 24 hours and add fresh medium? Is there any other method to stably overexpress mircoRNA in primary T cells?
Thanks in advance!
Polybrene at the concentrations used is likely to be toxic. Therefore, incubation with polybrene should be for a minimum time to facilitate viral infection for 3-6 h. If you can increase the number of viral particles in your assay, you do not need polybrene to increase your infection efficiency. Therefore, a reduction in time with polybrene may improve cell survival.
Hi Sipan, did you stimulate the T cells with CD3/CD28 and culure cells with IL-2? If you don't, the splenocytes tend to dead within two or three days even without any polybrene. For infection of lentivirus into T cells, you need to pre-activate cells with TCR and /or by cytokines inducing resting T cells to enter in G1b phase of the cell cycle because integration of the lentivirus genome do not occur in the resting T cells. Hope that helps.
Thank you, Qinglin! Do you have any experience in stimulating murine T cells by ConA? Does ConA have similar proliferation efficiency as CD3/CD28 in the presence of APCs? Does ConA lead to higher apoptosis after stimulation compared to CD3/CD28? Any advice will be much appreciated.
Hi Sipan, I never tried ConA, but ConA should have different response to CD3/CD28 because it uses different cellular signaling pathways to stimulate cells.
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