increased BrDU incorporation but similiar cell cycle profile by PI or DAPI - (Sep/13/2007 )

Hi, guys, I am just wondering how sensitive is PI or DAPI in displaying overproliferation (or G/S check point disregulation).
I have seen increased BrDU incorporation in the knock out cells vs the wild type cells. I also see decreased p21 expression in knock out cells. However, when I check the cell cycle status using either PI or DAPI, I did not see any difference for the percentage of the S and G2/M phase cells. Any ideas? Suggestions? I have seen some people using Hoechst and pyronin. Will these two dyes make a difference ?

Thanks a lot in advance.

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Hm, difficult...
Usually, cell cycle analysis by FACS is pretty sensitive, but you have to be careful to include enough cells in you analysis (the more the better obviously...), and to get the gating/settings right. You could try to play around with these...(well, probably you did already..)
I can not give you any advice concerning Hoechst and pyronin. I usually used PI, was fine. I was able to detect changes of about 1.5-2% (e.g.30->32%), which have not been visible in IHC.
A good friend of mine preferes EtBr, she sweares this gives better peaks.

Good Luck!
dedee

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Thanks a lot for your help.

Could you please tell me why the number of the cells matters? Say, I only have 10,000 cells but every tube contains the same number of cells, could I still get a reliable cell cycle analysis? Furthermore, have you ever fixed the cells with formaldehyde and then peameabilize and stain them with PI or DAPI. Could this give reliable results?

Thanks

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