You might try TO-PRO-3 or LDS-751 (from Molecular Probes).
This is the text from my Handbook:
Unlike the fluorescence of Hoechst dyes, the fluorescence of TO-PRO-3
(T-3605) and LDS 751 (L-7595) is considerably enhanced by the presence
of bromodeoxyuridine in DNA. In conjunction with propidium iodide, these
nucleic acid stains have been used to discriminate BrdU-labeled cells
from nonproliferating cells by flow cytometry and with an imaging
system for automated cell proliferation.
The relevant references on TO-PRO-3 are:
"Automated topographical cell proliferation analysis." van Raaij EJ, ten
Berge D, Hage W, Brouwer A, Meijlink F, Maintz JBA, Verbeek FJ.
Cytometry 45, 13 (2001)
"Fluorescence enhancement of DNA-bound TO-PRO-3 by incorporation of
bromodeoxyuridine to monitor cell cycle kinetics." Beisker W,
Weller-Mewe EM, Nusse M. Cytometry 37, 221-229 (1999)
Cytometry 1999 Nov 1;37(3):221-9
Fluorescence enhancement of DNA-bound TO-PRO-3 by
incorporation of bromodeoxyuridine to
monitor cell cycle kinetics.
Beisker W, Weller-Mewe EM, Nusse M.
GSF-National Research Center for Environment and
Health, Flow Cytometry Group, Neuherberg, Germany. beisker@gsf.de
BACKGROUND:The detection of DNA-incorporated
bromodeoxyuridine (BrdUrd) in mammalian cells is a well-known and
important technique to study cell cycle. The use
of TO-PRO-3 for detection of BrdUrd substitution of DNA by dual-laser
flow
cytometry has been investigated.
METHODS:Fluorescence enhancement of TO-PRO-3 in BrdUrd-labeled cells is
registered in
combination with the fluorescence emission of the
intercalating dye propidium iodide (PI) as a total DNA stain to give
bivariate
DNA/BrdUrd histograms. By the low concentration
of only 0.3 mircoM TO-PRO-3, BrdUrd detection is optimized, and
undisturbed
total DNA content by PI can be detected as well.
TO-PRO-3 is excited by a red HeNe laser and PI by an argon ion laser.
RESULTS:In order to understand the binding of
TO-PRO-3, energy transfer from PI to TO-PRO-3 has been measured as well
as
the influence of an external DNA binding dye such
as Hoechst 33258 with Adenine-Thymine (AT) binding specificity. Cell
cycle
studies of human SCL-2 keratinocytes and mouse
3T3 cells prove the method to be as generally applicable as the
classical
BrdUrd/Hoechst quenching technique, but without
need for expensive ultraviolet laser excitation. No BrdUrd sensitivity
could be
found for the similar dyes TO-PRO-1 and YO-PRO-3,
whereas TO-PRO-5 and YOYO-3 showed only very little sensitivity to
BrdUrd labeling as compared with TO-PRO-3.
CONCLUSIONS:Cell cycle studies of mammalian cells can be done by
dual-laser
flow cytometry without the need for ultraviolet
lasers by using the BrdUrd-dependent fluorescence enhancement of
TO-PRO-3.
Total DNA content can be measured simultaneously
using PI.
"Detection of bromodeoxyuridine incorporation by alteration of the
fluorescence
emission from nucleic acid binding dyes using only an argon ion laser."
Frey T. Cytometry 17, 310-318 (1994)
Cytometry 1994 Dec 1;17(4):310-8
Detection of bromodeoxyuridine incorporation by
alteration of the fluorescence emission from nucleic
acid binding dyes using only an argon ion laser.
Frey T.
Becton Dickinson Immunocytometry Systems, San
Jose, California 95131.
A method was developed that uses paired DNA dyes
to detect the incorporation of bromodeoxyuridine (BrdUrd) into cellular
DNA
and requires only 488 nm excitation. The
fluorescence of thiazole blue, TO-PRO-3, and LDS-751 was found to be
enhanced by
the presence of BrdUrd in DNA. Pairing LDS-751,
thiazole blue, or TO-PRO-3 with propidium iodide (PI) for flow cytometry
allowed the differentiation of cells containing
BrdUrd from BrdUrd unlabeled cells. LDS-751 can be excited directly at
488 nm,
and TO-PRO-3 or thiazole blue can be excited
indirectly by resonance energy transfer from PI. The enhancement of
fluorescence from these dyes is correlated with a
decrease in PI fluorescence, suggesting an increased energy transfer
from PI
to the red emitting dye. Fluorescence from other
dyes, including thiazole orange, TO-PRO-1, rhodamine 800, oxazine 750,
and
7-aminoactinomycin D, was not altered by the
presence of BrdUrd in DNA. Results also were obtained showing that
BrdUrd
detection using PI and TO-PRO-3 is compatible
with immunofluorescence staining with FITC-labeled antibodies.
andreas.simm@medizin.uni-halle.de wrote:
> Hello,
> I have a question of an user, which is not really
> related to folw cytometry. But as I have seen that
> there are from time to time questions about
> immunochemistry in this list, I want to forward
> his question.
>
> This user want to detect cell proliferation in
> tissue samples by immunohistochemistry.
> The method bases on detection of incorporated BrdU.
> He used a kit which costs so much, that he wants / needs
> to try an alternative.
> Do anybody knows a reliable method to detect
> incorporated BrdU by histochemistry?
>
> Thanks for your help in advance.
>
> Andreas
>
> PD Dr. Andreas Simm
> Universitaet Halle Wittenberg
> Klinik fuer Herz- und Thoraxchirurgie
> Ernst-Grube Str. 40
> D-06120 Halle
>
> Tel.: +49 (0) 345 557 2647
> FAX: +49 (0) 345 557 2782
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