Can an average enzyme signal from a lot of cells (n>25) have wide variation; with the normalized average being same (but with wide error bars) - be a due to different cell cycle or passage? Are there any control experiments for passage number?
Did you plate exactly the same number of cells on each of your experiment?
I agree with you Ian. Ismaeel are you working with primary cells or with immortalized cell lines (eg. With telomerase)?
Btw, aside from what Ian mentioned, you can do facs analysis for cell cycle of your cells and compare them.
In my opinion the best way is to keep checking the doubling time of the cells, with increasing passage number the doubling time is changed. If you find a significant change in doubling time start considering to throw those cells and revive the freezed cells of earlier passage.
I agree with Rahul, in addition to that the phenotype of cells get changed in later passages they look streched more elongated than earlier passage cells. Its better you write passage number, date, cell type on your flask to avoid this problem in future.
Dear Ismaeel, I also have problem like you regarding different passage of cell giving a different analysis result . I assume in your case, the were different of enzyme level in different passages of same cell. However, mine there are different result in 'gene level' of different passages of the same cell. I agree with Rahul and Mairaj point, in my case, cell which have change in doubling time will given a different result of gene copy number. As a solution, I will make sure that only cell with similar doubling time being proceed with gene copy number analysis.
thank you fellow researchers and advanced Scientists. the recommendation on checking telomerase shortening, synchronization, and using fresh cells are well received.
i use fresh cells, they are morphologically similar (high cytoplasm to nuclei ratio; well spread and normal as depicted in ATCC); and treat with reduced serum (starvation -for synchronization) but still i got varied results. i guess there are other factors in the assay that are time/conc/stimuli specific.
if it doesnt work, i guess i need to do the telomerase experiment which is not that easy. no wonder it won the noble prize.
thanks again.
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