I used to transduce CD3CD28 bead stimulate PBMC with Lentivirus. In the first few days after transduction, PBMC show GFP. I keep adding hIL-2 every other day. But perhaps a week later, GFP signal is almost gone, and PBMC grows slowly. Some of them died too. How long maxi can PBMC be kept growing healthy with GFP signal? How to do that. Do I need to add something else, like proleukin? Thank you
Theoretically, GFP dose not influence on the phenotype of transduced cells, at least in our experiment. The transduced T cell usually can be alive for more than 3-4 weeks
I have a quick question. what is the amount of CD3/CD28 Beads you use for pre-stimulate PBMC as well as the amount of hIL-2. Too strong stimulation may exhaust T cells. But it happens that PBMC from different donor may have different character.
Besides, how did you perform T cell transduction? If you still observe too many cells died even tried a lot of optimization, you may think about concentrate virus instead of using rude viral supernatant, try RD114 psedotyped virus to replace VSV-G(high titer, but may harm to the cells)
Hope that helps
Best
Linchun Jin
simple tips may be helpful.. using beads or other stimuli will give stress to the cells , no matter what kind. beads are good, better to use coated antibodies... usually transgene will not go after few weeks of culture but you should freeze them once they are convincingly expressed and reactivate them for your other experiments... remember do not stress them. too much IL2 will kill them as well, dirty and quick spin or split will kill and also keep APC to be company with T cells. CD4 is more fragile and CD8. good luck
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