I would like some advice regarding my experiment. I am using a transfection protocol to make viruses with my HEK293T producer cells. After I waited the required 48hrs, the produced virus (supernatant) is harvested and concentrated, but when used to infect cells like HEK cells or Primary astrocytes, they didn't seem to work. Our protocol was working before, but now it seems not to work, and I am unaware of the possible reasons. I see very reasonable transfection efficacy, so I wonder why I barely see any infection when I use the virus produced to infect.
In summary, my ratios (per 10cm plate) are currently 20ug for transfer plasmid, 15ug for pD8.91, 6ug for pCMV-VSVG(envelope). I incubate in DMEM+FBS without PS overnight and transfect with PEI. I change the media after about 12 hours to DMEM+FBS with PS
I can provide more information as needed. Any advice on why my harvested virus seems to produce no infection/transduction would be appreciated
Did you calculated the titer of your lentivirus. Titer determination would help you to understand the MOI you should use for the transduction and by using higher MOI you can achieve the good transduction efficiency.
Is that the same cell line that you used before?
You may use more plates to collect the viruses, may be using higher MOI will help you to increase the transduction efficiency.
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