I did transfection using lipofectamin 2000 to get stable GFP positive cell line, but after transfection and puromycin selection my HUVEC growth rate reduced. I have not seen any colony displayed cell proliferation even after 14 days in 96 cell culture plate. However, cells has still displayed green flourescent . Has anyone experienced this, and would this be of concern? I appreciate your comments.
Transfection is very stressful for cells so it is normal that some cell line growth rate reduces dramatically after that. You might want to optimize the amount of cell you are seeding per well in different ratio.
Have you checked kill curve for your cells for puromycin. And other is the concentration of lipofectamin used in experiment, as it is toxic for cells so try optimizing lipofectamin concentration or use less.
Thank you son much. I have checked the optimal concentration of puromycin for my cells, I added puromycin just for one day in cell culture medium. For lipofectamin concentration, I followed the protocol, using the low concentration about 0.6 microliter/ml in a 96 well-plate culture. I have tracked cells that expressed GFP for up to 14 days. While GFP positive cells displayed good fluorescence, there is no sign of proliferation. I do not know if I keep them for more than one months, will I see any colony formation?
You might try keeping them for more than a month but I do not think that will bring any change on the rate of proliferation.
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