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> We have recently been studying the mechanism of some novel oncolytic agents
> in my laboratory and have observed an inconsistency in some acquired flow
> cytometric data. When the novel compound is run in a typical propidium iodide
> stained cell cycle analysis , an obvious strong accumulation of S-phase cells
> is observed. When the same compound is run at the same concentrations
> (etc......) in a BrdU incorporation/antibody flow detection method, no such
> S-phase accumulation is observed!
> Can anyone give me some possible explanations for what is really happening
> here? Can there be structural changes in DNA that mimic S-phase accumulations?
> Which method is a more reliable indicator of the effects noticed?
Given the fairly severe denaturation of DNA required to give antibody
access to incorporated BrdU, I'm not surprised that some cells could be
affected. You could show that S-phase accumulations are indeed detectable by
BrdU incorporation with either the BrdU quenching of Hoechst, or the very
elegant method of Crissman and Steinkamp (BrdU Hoechst quenching combined
with an unquenched DNA fluorochrome). Neither method requires antibody
binding. The Crissman and Steinkamp method gives similar looking bivariate
displays of anti-BrdU vs. DNA; the electronic processing can be done in
software.
Details of anti-BrdU observations you could also get from Terry Fetterhof at
Boehringer-Mannheim up the Interstate from you.
Dave Coder
Cell Analysis Facility
University of Washington
tel. 206-685-3014
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