Dear researchers,

I have been trying to transfect the HCT116 cell line having p21 knock-out, which makes these cells less able to attach and more sensitive. Despite having no problems with cultivating them in larger flasks, when I try to plate them in a 96-well plate and transfect them, even my controls (w/o transfection mix, just medium) start dying rapidly within 24h of transfection. I tried coating the plate with poly-d-lysine before plating cells, but I saw little to no improvement. I tried transfecting parental HCT116 using Lipofectamine 3000 and 2 and 0.5 micrograms of my plasmid and it worked just fine. With p21 -/- cells, however, it failed, so I tried adding less pDNA (0.1 micrograms and 75 nanograms) and had the same results. I also tried the ScreenFectA reagent protocol for cells which were already plated for 24h and also had the same issues. I always try to be as gentle as possible to avoid detaching the cells. What would you suggest doing to improve adhesion and increase cell survival after transfection? I need to keep them alive for at least 48 hours so I can use them for other experiments. Transfection itself worked well, I had a lot of transfected cells, but many of them were dead within 48 hours. Any tips are highly appreciated.