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Dear Gil,
From your account, a cell cycle analysis by triple staining flow cytometry
will probably solve your problem: 1-DNA (with 7-amino-actinomycin to stain
for total DNA), 2-BrdU incorporation (to identify S-phase cells, usually
FITC-anti-BrdU Ab), 3-Your phenotypic marker (to gate the cells of your
interest). Critical points are that DNA MUST be analyzed in a linear scale
while your protein of interest could/should be analyzed in the log scale.
Let me know if it works for you,
Best,
Oscar
Oscar Sierra, M.D.
Div. Bone & Mineral Diseases
Washington University Sch Med
Campus Box 8301
660 S. Euclid Ave
St Louis, MO 63110
-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]]On
Behalf Of Guillermo Palchik
Sent: Wednesday, December 08, 2004 4:45 PM
To: [log in to unmask]
Subject: G2 Stage - FISH?
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Dear Confocalists,
I am trying to find out some information for a
colleague. She wants to find out whether a particular cell is arrested in
the G2 stage. There is a special population of cells in the (rat) mammary
epithelium that represent about 18% of the epithelial cells. These cells
have very large nucleii and they have shown that these cells undergo
mitosis in vivo.
Others have demonstrated that they also mitose in vitro,
however, they do NOT synthesize DNA before they do so! In vivo, 13 to 48%
of these cells are PCNA positive depending on the stage of estrus making
52 to 88% PCNA negative (depending on the estrus cycle). It is because of
these characteristics, that they believe that these cells may be arrested
in G2, and if so, they would have double the normal complement of
chromosomes. To test this hypothesis I would like to stain with a marker
for a particular chromosome - any chromosome. However, I am concerned
about the fact that I would not be able to easily quantify their amount. I
must say that I was only presented with this problem yesterday and am
still in the process of researching the subject. I don’t know if I can use
FISH for this and I am not very knowledgeable on its applications (more
than the theoretical principles). So, my question is: would FISH help me
solve the problem? If so, how do I set up a FISH experiment (do I need to
have special hardware?) Should I target other proteins instead (cdc25,
etc.)? Any suggestions would be greatly appreciated.
Best Regards,
Gil Palchik
Manager - Light and Confocal Microscopy
Microscopy and Imaging Shared Resource
Georgetown University Medical Center
Tel: 202-687-1916
Email: [log in to unmask]
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