RE: Cell cycle, BrdU incorporation, and Apoptosis (TUNEL)

There are numerous methods that allow one to define in which cell cycle
phase cells are dying by apoptosis. The most widely used is TUNEL assay.
We reviewed these methods many times, - e.g. in Methods in Cell Biology,
Vol 75 (2004) Chapter 12: "Cytometric Methods to Detect Apoptosis". It
is even possible to define whether G0 or G1 lymphocytes undergo
apoptosis by measurement of cellular DNA and RNA content after staining
cells with acridine orange  (e.g. see Huang et. al. Cytometric
assessment of DNA damage in relation to cell cycle phase and apoptosis.
Cell Proliferation, 38: 223-243, 2005.)


Zbigniew Darzynkiewicz, M.D., Ph.D.
Brander Cancer Research Institute 
New York Medical College
Valhalla, NY, 10595

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



-----Original Message-----
From: Matthew Janes [mailto:mjanes@uci.edu] 
Sent: Wednesday, June 21, 2006 3:04 PM
To: cyto-inbox
Subject: Cell cycle, BrdU incorporation, and Apoptosis (TUNEL)

I want to determine in what stage of the cell cycle particular 
primary lymphocytes are dying.	So ask the community if anyone has a 
recommended protocol for CELL CYCLE (DNA content with BrdU 
incorporation to properly distinguish all cell cycle phases) with 
TUNEL, or another well established method for assaying for apoptosis.

Or can sorting of populations in the different stages of the cell 
cycle be performed?  I am experienced with sorting, so is it a 
problem sorting different populations when aquiring data on a linear 
scale (like when using PI, 7AAD, or DAPI for cell cycle?   Would the 
community recommend just sorting different lymphocytes based off 
their cell cycle, the subsequently enumerate apoptosis by microscopy, 
or look at the degree of DNA fragmentation on a gel.

Any opinion on such a task would be greatly appreciated.

best,

Matt
-- 
Matthew R. Janes, MSPH
Ph.D. Candidate
Department of Molecular Biology & Biochemistry
Center for Immunology
University of California at Irvine
mjanes@uci.edu