GFP and BrdU staining
hms at shapirolab.com
Mon Feb 24 11:21:08 EST 1997
>Does anyone have experience staining GFP (S65T, humanized codon, not a
>fusion protein)-expressing cells for BrdU incorporation? I'm afraid
>standard alcohol fixation and HCl treatment is too harsh for GFP. I
>read about nuclease treatment and UV breaking. Which anti-BrdU
>antibody would you recommend?
The UV breaking method, about which you should seek more information from
Darzynkiewicz et al, does not require anti-BrdU antibody, and may be doable
under gentle enough preparative conditions to preserve your GFP.
Cytochemical methods of BrdU detection, based on staining DNA with one dye
which is quenched by BrdU (originally Hoechst 33258, but Tom Frey at B-D,
among others, has described alternatives) and one which is not, could be
used with gentle fixation and/or permeabilization, but are probably less
sensitive than antibody- or strand break methods.
>In fact, is there a better way to detect proliferating cells? Is
>there, say, a thymidine analog that is incorporated into newly
>synthesized DNA and fluoresces on its own?
BrdU and tritiated thymidine are the gold standards. There are
intrinsically fluorescent thymidine analogs which can be incorporated into
DNA, but they require excitation at around 260 nm, which is highly
impractical for almost all flow cytometers, and, as I recall, their quantum
efficiency isn't all that high. You could use tritiated thymidine if you
sorted the cells of interest onto a slide for subsequent autoradiography.
>antigen that is "really" specific to some cell cycle stage?
Ki-67 and the various cyclins have been used, but haven't yet achieved gold
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