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Position: Discussion forums > Bioscience biotechniques discussion forums > Cell biology and histology methods forum
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cell cycle
Posted by: andreadn (IP Hidden, New member, 2)
Date: May 16, 2005 05:03PM
I want to determine the cell cycle length of fibroblasts, I followed the BrdU/PI protocol, giving a 30 mins. pulse with BrdU, washing and then harvesting each hour, then reading with flow cytometry. I have samples for 7 hours and am analyzing with WinMdi and FlowJO. I havenīt been able to determine the length of the cycle, my histograms are very strange, and have very high percentages of cells in S phase (up to 55%). Flowjo canīt determine the cell cycles with the DNA content that I have. Do I have to collect cells during more hours, and is it normal to have those percentages?
Re: cell cycle
Posted by: huangz123 (IP Hidden, Junior member, 19)
Date: May 16, 2005 11:14PM
Have you synchronzed cells in G0/G1 and then release them into cell cycle? Cells normally will only be synchronized for one cycle and will lose synchronization soon after. It seems your cells are very proliferative. I guess you also counted BrdU positive cells using immunocytochemistry staining.
Re: cell cycle
Posted by: 5'-ATCG (IP Hidden, Unregistered user, )
Date: May 17, 2005 03:45PM
First thing is first. Do cell growth curve to determine the doubling time of your cells in your condition, from there determine your window for flow cytometry. Serum starvation or DNA synthesis inhibitor are very common ways to synchronize your cells.
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