Hi, i'm desesperate. i'm trying to produce lentivirus usinf BEI resources kit NR-52948.
24 hours after transfection i detect a massive GFP expression in my 293T cells.
After 72 hours i filtered (0.45um) and infected VERO E6 cells. well! not even a single cells infected express GFP after 48-72 hours.
I also tried to combine BEI plasmid NR-52516 (Luc-zsGFP) with pCMVdR8.2 and NR-52514 (plasmid expressing cov spike). nothing! then i tried using psPAX2, NR-52516
(Luc-zsGFP) and an alternative VRC8400 expressing the Cov-2 spike and alternatively NR-52514. Again nothing. I don't know what i'm doing wrong. I have absolutely zero GFP positive cells after infecton. please support.
Have you checked in flow cytometry? I guess that most flow cytometers are more sensitive than microscopes and the human eye. A wild guess is that not enough of the transfer plasmid is in the mix and therefore not enough is delivered and expressed?
Using "Luc-zsGFP", you may also switch to a luciferase assay? This may help.
Perhaps an ACE2-overexpressing cell line may help to make sure that there is plenty of the "receptor". Do you have a control pseudovirus such as the traditional one using VSV-G?
Hi. Usually florescence microscopy is enough to detect positive cell. If is not visible as fluorescence i don't consider as positive infection. Vero cell are highly and easily infectable as i assessed previously using a lentivirus i purchased. My only conclusion is or the virus I'm producing are not working or not virus is produced.
So, you have no signal above background in flow? My best guesses are that the titer is low, because packaging does not work well (the ratios of the plasmids may have to be adjusted?) or 293T cells are too old.
We use a 293ACE2 transfected cell population to transduce with pseudovirions. Seems to work in these 293ACE2 cells than in Calu3 and Vero cells for both the control and the Spike virus.
On a good day after 24 hours under the microscope, probably even faster. However, most of the time it requires flow cytometry to "see" the GFP signal. The control virus is about 10-100x brighter depending on the virus prep.
Is the signal so faint? With the virus i bought from bps i could see perfectly under fluorescence microscope. The signal was bright.
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