Hello,
I did a general comparison of cell-cycle analysis between my protein/CNT overexpressed HEK cell poupation. However, after 24hour of transfection, it shows a 10% increase in S-phase whereas after 48 hour of transfection, there is exact 10% decrease. Is there anyone know how to explain this data?
Any insight would be helpful.
i would be highly cautious with hek cells..
better to use other cell lines...for CC analysis
Hi Didier,
I was aware of the point and in fact I asked the same question here also but HEK seems to be a standard cell line for CC analysis as per many literature and several people assured me to go with the experiment, so I did it with proper control and conditions.
being an old CC researcher..I do not agree "the HEK are worth using for CC analysis"..i am using these as a test tube...
they are not good for imaging, heavely transformed , they easely detach and so on.
see this paper for an historical view of 293T
DOI: 10.1038/ncomms5767
Genome dynamics of the human embryonic
kidney 293 lineage in response to cell biology
manipulations
Hi Didier,
Any specific recommendation then? which cell line do you think has good transfection efficiency as well as suitable for CC analysis?
Although I agree with Didier about the use of HEK293 cells, maybe your result tells you that overexpressing your protein somehow partly synchronizes the cells, stimulating entry into S-phase, then accumulating in G2/M for example, which would explain why more cells are in S phase, then less cells are.
It deeply depend about your work..many more or less transformed cells lines are easely transfected , currently using U2OS..and got routinely 60-80% with turbofect reagent. (2.10e5 cells seeded/ 6wells plate 2,5ug DNA/4ul Turbofect TF 2hours on the cells. or 1.10e6 cells for 10cm plate/8ug DNA/16Ul TF .
hope this help
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