Re: Continuous BrdU labelling-Hoechst 33258 assay

Peter Rabinovitch (petersr@u.washington.edu)
Tue, 16 May 1995 11:27:09 -0700 (PDT)

Sorry that the basis for the relative extent of quenching in sucessive
cell cycles has not been made clearer. The initial Hoechst fluorescence
drops by 60-70% as a result of BrdU quenching. This DNA has unifilliary
substitution by BrdU, and because high BrdU concentrations are used, a
high proportion of Ts are replaced. This extent of quenching is a large
proportion of that which can be obtained, even in totally BrdU substituted
DNA. In the next round of DNA synthesis, half of the DNA becomes
bifilliary substituted as well; however, the difference between the degree
of Ho quenching between unifilliary and bifilliary substitution is just
not as great as the difference in fluorescence between unsubstituted and
unifilliary substututed DNA. Thus, the additional increment of reduced
fluorescence is modest. In the next round of DNA synthesis, 3/4 of DNA has
BrdU in both strands, but again, the difference between this and the
previous cycle is modest, usually just barely enough to permit separation
in the Ho/EB plot.

I hope that this is somewhat helpful.

-Peter Rabinovitch

On Mon, 15 May 1995 Yashpal_Agrawal@cis.ucsd.edu wrote:

> This is in reference to the continuous Brdu labelling-Hoechst
> 33258 assay (described by Martin Poot, P.Rabinovitich,
> M.Kubbies) for studying 3 consecutive cell cycles.
>
> In the assay the Hoechst fluorescence is quenched by Brdu,
> which results in a decrease in Hoechst fluorescence of S
> phase cells during the first cell cycle.
>
> What is unclear to me is: Why does the Hoechst fluorescence
> of S phase cells increase when cells are in the second and
> third cell cycles ?.
> In the available literature (Methods in Cell Biology, vol
> 41), the authors suggest that this has something to do with
> single or double strand labelling.
>
> Could somebody please explain ?
>

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