RE: S-Phase marker

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).

 

 

Zbigniew Darzynkiewicz, M.D., Ph.D.

Brander Cancer Research Institute at NYMC

19 Bradhurst Avenue, Suite 2400

Hawthorne, N.Y. 10532

darzynk@nymc.edu

http://www.darzynkiewicz.com/zbigniew/

 

-----Original Message-----
From: Facslab [mailto:facslab@uni-mainz.de] 
Sent: Friday, December 09, 2005 5:06 AM
To: cyto-inbox
Subject: S-Phase marker

 

Hello Flow community, 

 

I have a phd student with following question. 

 

Thanx in advance 

 

Steffen 

 

Here is the question: 

 

Hello Flowers, 

 

I'm a PhD-Student at the Institute of Toxicology at university in Mainz, germany. 

 

I have a short question: 

I work with human cancer cell lines by treating with DNA-damage agents. Before the cells undergo Apoptosis I observe a late S-Phase or a G2/M-Phase cell cycle block by Propidium Iodide (PI) Staining. 

Now I'd like to discriminate the S-Phase and G2 or M-Phase using a PI Staining combined with a specific S-Phase marker (perhaps a specific S-Phase protein). 

Has anybody an idea witch marker is useful for this kind of discrimination? 

 

Thanks very much Steffen Naumann 

 

 

=============================== 

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 

 

www.facslab.toxikologie.uni-mainz.de 

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