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Transfecting cells for cell cycle analysis, how to deal with cell density?

Transfection Cell Cycle Ethanol Fixation Propidium iodide stain Cell Density

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#1 Epigeneticist

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Posted 28 July 2014 - 07:53 PM

I understand that cell density plays a major part in cell cycle analysis, specifically having the cells in log phase growth prior to fixing them. How do you deal with cell density issues when transfecting cells, especially when confluency needs to be high (i.e using lipofectamine)?

 

I was planning to transfect (express construct or siRNA) mammalian cells in a 6-well dish at 80-90% confluency then split cells to a 60mm or 10cm dish 24-48hrs post transfection then fix cells for cell cycle analysis at 72hrs. I was thinking that splitting the cells to a lower cell density for the final 24-48 hrs would be the best approach. 

 

Has anyone done something similar? Recommendations please. 

 

Thanks!



#2 seanspotatobusiness

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Posted 04 September 2014 - 05:59 PM

I've not done any cell-cycle analysis but in my experience expression of transiently-transfected plasmid peaks around 72-hours post transfection so I guess 72-96 hours post transfection would be a good time to fix the cells.







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