RE: Annexin V/Propidium Iodide Staining for Adherent Cells

Hi Brandon,

It is well known that removal of adherent cells for (flow) analysis creates all kinds of
sample integrity problems therefore it is highly advisable to analyze adherent cell lines
in their native environment. There are a number of options available (or more correctly
can be available) to you other that flow techniques (solid phase CCD based systems, laser
and PMT-based systems). They all have their Pros and Cons but definitely are worth
considering when planning an adherent cell experiment. I am particularly biased towards
LSC (laser scanning cytometry technology) because a) I represent CompuCyte as a
commercial entity; b) most importantly, performance characteristics of the LSC systems
match those of flow cytometry instrumentation in terms of precision, dynamic range and
sensitivity with identical data presentation but allow to simultaneously produce
excellent image data on SOLID phase specimens (adherent cells and tissues).

More specific references relevant to Annexin 5 staining and/ or LSC technology are
provided below:

1. The DNA of Annexin V-binding Apoptotic Cells Is Highly Fragmented, Zsolt Bacsó,
Richard B. Everson and James F. Eliason, Cancer Research 60, 4623-4628, August 15, 2000

2. http://www.compucyte.com/pubapoptosis.htm

3. J. Biol. Chem., Vol. 279, Issue 37, 39075-39084, September 10, 2004

Elena Holden,
CompuCyte Corporation
www.compucyte.com


-----Original Message-----
From: Tavshanjian, Brandon [mailto:Brandon.Tavshanjian@ucsf.edu] 
Sent: Thursday, February 07, 2008 5:35 PM
To: cyto-inbox
Subject: Annexin V/Propidium Iodide Staining for Adherent Cells

Our lab has used a protocol for FITC-annexin V/PI staining to look at induction of
apoptosis by flow which seems to work quite well in suspension BaF3 cells.  I'd like to
use this technique in adherent cells (MCF7,MCF10A, other mammary epithelial cell lines),
however, I've heard there are some issues with applying this technique to adherent cells
(namely, that the cell detachment procedure may cause false-positive staining by annexin
V).  I was wondering if anyone has a protocol like this that they've successfully used
for adherent cells?

------------------------------------------------------
Brandon Tavshanjian
UCSF Chemistry and Chemical Biology
Shokat Lab, Genentech Hall N-528
(415) 514-4111