I would like to troubleshoot the transfection stage of my lentiviral transduction protocol. In short, I have 4 lentiviral plasmids, one of which encodes GFP. I tried transfecting the HEK293T cells, harvesting the lentivirus and transducing the Jurkat cells afterwards (as per established protocol) but I had no GFP detected by flow on the Jurkats. Before doing any more transductions, I would like to check whether my transfection is actually working. I’d like to do this by transfecting the 293T cells with the same plasmids again and running the cells on flow cytometer to check for GFP. Can I actually do that though if the cells could potentially be producing the lentivirus post-transfection? Would fixing the cells before running on flow stop the lentiviral production? I know that you can use flow to detect GFP post-transfection but is it safe to do this if I am dealing with lentivirus production ?
Thank you very much in advance for your help.
How long did you wait for HEK293T cells produce lentiviruses after transfection. Usually it takes 72 hrs to see the GFP signals in HEK293T after transfecting. I would try to see the GFP signals under the microscope briefly before starting Flow, just to make sure if I am seeing the green signals. If you cannot see the GFP signals after 72 hrs, then your infected Jurkat cells probably won't have GFP signals either. It will not drastically reduce the GFP signals if you fix the cells, and immediately examining the cells. After fixing your cells, I think you can run through flow because your cells would have been washed with PBS, Fixation solution, PBS again before the flow. But if your lentivirus titer is low, you may can concentrate it with ultracentrifuge too.
Maria Sharif I think it is safe because you are using 4 plasmids for packaging and it is very safe. on the other hand, packaging of the virus with 4 plasmids does not seem to be as short as flow cytometry. for flow cytometry, the samples are trypsinized, then washed with PBS 3 times (the supernatant containing viruses is removed), and read within 30 min to 2 hours.
note: a good transfection (high GFP+) does not mean to have a high titer virus and good transduction; this just indicates that your GFP+ vector (not the other vectors) is well transferred to the cell line.
Thank you very much everyone for your answers, that’s really helpful! In the protocol I have the lentivirus is harvested from the 293T cells 48 hours post-transfection. I know other people do 72 hours as well though so might try that!
- Badges
- Science method