i need to ask that when I transduce my HEK293T cells with Lentivirus, how much time will they require for transduction because when I add lentivirus and then change the media after 24 hours, my cells condition is too bad they they don't grow when I passage them? i have repeated this transduction 3 times. and one thing more if I succeed in transduction and then want to start selection in puromycin media how much concentration of serum I should use in that media 2% or 10 %. and for how much passages ( to confirm for a stable cell line)
lentiviral transduction depends on the MOI - multiplicity of infection, how many virus particles/cell you are adding. perhaps you are adding to much virus and it is overwhelming the cells. try an MOI of 1, 2, 5, and 10. see which cells look the healthiest. you could also choose one MOI and remove the virus after different time points.
transduce your experimental cells at the optimal MOI/time and let them grow out some and then add puromycin with 10% FBS along with untransduced cells with puromycin with 10% FBS. Monitor the untransfected cells for death to determine when your transduced cells are ready. you can maintain puromycin at a lower concentration to keep selective pressure on.
Puromycin concentrations have to be determined empirically for your cells in your hands. test different concentrations on untransduced cells and use the one that kills the cells in the selected time frame - you should be able to get this information from a literature search for your cell line.
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