RE: Annexin V/Propidium Iodide Staining for Adherent Cells

In principle, I agree.	However:

It would be fairly straight-forward to develop apoptosis assays for adherent cell lines,
provided that ample QC were done to characterize the "damage" done by adherent cell
harvesting.

In my experience, many adherent cell lines lend themselves well to apoptosis assays by
flow, provided that care is taken in the harvesting protocol.  If the harvesting is done
properly (for many, NOT ALL adherent cell lines), then the impact on the quantification,
etc.,  of apoptotic cells is minimal.

If this involves using non-flow based instruments/techniques, well, that's just good
science.  Many assays have "evolved" past the point of needing PCR, western blot, etc.,
to corroborate their results, but backing up flow data with images and/or molecular or
other data is essential to successfully using flow assays in research and publishing.

Many scientists who bring samples to a flow facility would be surprised at how much
information can be gleaned from simply taking a brightfield picture of their cells.

Andrew

Andrew Beernink
Senior Applications Specialist
Beckman Coulter
(858) 353-7007
(858) 435-1137 efax


-----Original Message-----
From: Elena Holden [mailto:EHolden@compucyte.com] 
Sent: Friday, February 08, 2008 3:56 PM
To: cyto-inbox
Subject: 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