recently when packing lentivirus , my 293t lose attachment to the plate after being transfected and the medium became very very yellow. Even more , the status of target cell after being infected with the virus harvested this time became very bad. Firstly , I think maybe there is some problem with my reagent. SO I CHANGE THE CACAL2 AND 2X HBS. But it doesn't work.
Usually 293T detach from the surface and medium gets yellow when they are too confluent. Do you count your cells before preparing your transfection plates? Other explanation could be that your lentivirus is toxic both to 293T and to your target cells. In this case maybe try to transfect less DNA and collect your lentiviruses faster that usually. Finally, yellow medium can be a sign of contamination, but you would definitely see that under the microscope.
thanks a lot for ur advice. Except the last posibility , others can be excluded . But i didn't observe any bacterial. Now i doubt it might be contaminated by mycoplasma.
Do you know what may cause this kind of contamination ? thanks a lot
Hi Gong,
I assume that your amplifying a lentivirus pseudotyped with VSV-G envelope. Cells expressing this viral envelope tend to fuse forming poly-nucleated cells that die shortly after transfection.
Here are my recommendation: First, review your ratio of plasmids DNA. Ideal ratio is 1:1:1 (Shuttle:evelope:genome). Second, make sure the cells are in exponential phase and no more than 70% confluent at the time of transfection. The cell division will not dilute the plasmid (Cell contain the Large T antigen that allow for replication of DNA of procaryotic origin).
As for the contamination, you will be able to see if there are bacteria under microscope. If any of your reagents is contaminated. This this possibility by mixing reagent with media (separately) with cells and incubate at 37' like you're growing cells. However, if you think you have mycoplasma (BTW, you can't see them on regular microscope) you can test specifically for that, there are many PCR-based detection kit available. The infection tends to be chronic and cell just don't look healthy (cell detaching, large vacuols..). If your cells are contaminated with mycoplasma, just bleach them, another batch.
Good luck
Hi, My cells have died after transfection, but the media did not turn yellow. We have realized that lipofectime can kill mycoplasma infected cells.
thanks a lot for ur kind help. Now i am repeating this EXP to find what the problem is.
i change all the regent that can be replaced . thanks lot
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