I'm trying to do a lentiviral transfection/transduction of U87 glioma cells. I transfected my vector into HEK283FT cells (packaging cells) and I wanted to use the viral media on the U87 cells. The U87 cells were growing fine but they all died when I did the infection. I'm aware that U87 cells are sensitive to polybrene so i tried it with and without polybrene and all the cultures died.
I've done the same infection with glioma stem cells and those have been fine so I'm not sure what's going on. Anyone have any suggestions or a protocol specifically for U87 transduction? Thanks!
Hi Manjula- thanks for your response. I only did my transfection in low serum media. The actual transduction was with serum. Other people in my lab had told me about the sensitivity to polybrene- that's why I tried it without, but I still ended up killing my cells.
As for the gene, it's a microRNA, but i'm not aware of it inducing apoptosis. I can always try it with a control vector to compare, but I'm doubtful that the cell death is due to the gene itself. Thanks again!
Hi,
If you have a doubt about the effect of your miRNA, you have to design a control. In any case, when you used miRNA you have to do controls with another LV which express reporter (transduction control) and a LV scramble (miRNA effect control).
Another point is that you could used LV after concentration and purification. In this way you will have a better efficiency (glioma cells are not the easiest cells to transduce) and you avoid potentiel problems about the compatibility of the medium between HEK and U87
Nicolas
Thanks everyone! I wanted to initially just do a test run with just my miR-of-interest, but I'll try a control vector too just to make sure that it's not a problem of apoptosis-induction due to the miRNA.
As for the medium, we use the same media for both our lines and they both grow fine in it, so I don't think that should be an issue. I'll try out the controls and hopefully that'll be the end of my troubleshooting!
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