Summary Document

This document is a summary of message from the Purdue University Cytometry Laboratories Cytometry Discussion list.

http://www.cyto.purdue.edu/hmarchiv/index.htm

Dr. Steffen Schmitt
FACS und Array Core Facility
NMFZ- Johannes Gutenberg Universität
Obere Zahlbacherstr. 67
55131 Mainz

Tel.: +49 6131 39 30219
Fax: +49 6131 230506

http://www.facslab.toxikologie.uni-mainz.de
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Dear flowers,

late but nevertheless. Here are the answers to our question to
discriminate S phase from G2/ M phase. A heartful thanks to all, who
had responded.

Steffen

Here are the answers:
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1)
You can use BrdU staining combined with PI to monitor the
proliferation of your tumor cells after treatment. BrdU will allow you
to discriminate between S-phase and G1 or G2/M cells. You can also make
use of phosphohistone-3 which is expressed only during mitosis (G2/M).
Upstate (upstate.com) sells an anti-phosphohistone-3 antibody for the
detection of this mitotic cell marker.
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2)
As a marker of un-blocked S-phase cells I would suggest a short pulse
(10-15 min) of BrdU followed by detection of BrdU incorporation by
standard methods (e.g. Dolbeare & Selden, Meth Cell Biol Vol 41,
298-317, 1994). If the cells were blocked in G2, the S-phase cells
would still be incorporating BrdU, if blocked in S – there will be no
BrdU incorporation. The alternative approach may be detection of the
detergent-insoluble fraction of PCNA. Cell fixation in methanol
combined with extraction of detergent-soluble fraction of PCNA leaves
only chromatin-bound PCNA, which when detected immunocytochemically, is
a good marker of S phase (The method is described by Larsen et al.,
Meth Cell Biol., 63:410-431, 2001). However, after some damage to DNA,
PCNA may aggregate in nuclear foci and not be extractable by detergents
regardless of the cell cycle phase.  Expression of cyclin B1 may be
used as marker of G2 cells – (e.g. Methods Cell Biol., 41: 421-435,
1994). One has to be careful, however, using cyclins as markers of
cells cycle position, because in some tumor lines, or as a result of
cell treatment with the cell cycle-perturbing agents, their expression
is unscheduled, i.e. cyclin B1 may be expressed also in other than G2
cell cycle phases (e.g. Gong et al., Cell Growth Differ.
6:1485-93,1995).
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3)
Anti-BrUdR antibodies would help distinguish early s-phase from G1 and
late s-phase from G2. This is used in combination with PI. Check Becton
Dickinson for the product..
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4)
I'd recommend using aBrdU-FITC to stain and separate out your S-phase
cells, along with using your current PI protocol.
Good luck...
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5)
You can do BrdU uptake for the DNA and stain with histone H3 for
mitosis, the trick is to use the "BrdU flow kit" that uses DNAse to
expose the BrdU inserted into the DNA and since you skip the HCL
treatment you can stain other proteins as the phospho histone 3 for
mitosis.
See becton dickinson cat# 559619. I have done proliferation and
apoptosis using this kit and p85PARP from Promega and it works
beautiful.
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6)
No need of another marker, just look carefully to technical adjustment
of your run

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