Apoptosis, Annexin V and Ficoll

Mark Kukuruga kukuru at med.umich.edu
Thu Mar 22 18:19:31 EST 2001


Hello all,
First, a general comment . . .  saying that annexin V labeling without a viability
indicator is useless (even misleading) is a bit too strong.  Cells that are "dead" at the
start of the apoptosis induction (assuming there is such an event) may not have become
so by apoptosis, and it's important to be aware of this . . . i.e., this contributes
to noise in your assay.  However, this may not be important for all applications,
where one, for example, might be concerned with total apoptosis regardless of time.
Adding a viability marker here does make the  measurement cleaner, and perhaps easier
to interpret.  Regardless, it is important to know that detecting apoptosis by annexin
V must be validated by a companion method, and everyone should first detect apoptosis
by morphology as their first step.

Peter,
Regarding your questions:
1)  While Ficoll separation may eliminate much of the "dead" population prior to
labeling, I would still expect some Annexin+/PI+ cells.
2) Yes, as apoptotic cells shift in their light scatter properties.  This shift is
a time/progression dependant process, so position of apoptotic cells in the FS/SS
distribution may be difficult to predict.  Correlating your apoptosis measurement
parameter with light scatter parameters can help with this.
MAK.


--
Mark A. KuKuruga, Managing Director
University of Michigan Flow Core
7416 CCGC 0946
(734) 647-3216, fax (734) 936-7376
kukuru at umich.edu


>>> <PLopez at adarc.org> 03/21/01 05:02PM >>>

Hello,

I have two questions related to the Annexin V apoptosis thread:

1- In human PBMC preps, would the Annexin V (+), PI (+) fraction
go down the drain if the prep was made using a Ficoll separation?
2- Is the Annexin V (+), PI(+) fraction something that might be
unintentionally gated out if the light scatter gate is too tight ?

Peter Lopez
The Aaron Diamond AIDS Research Center
212.448.5188 (office)
212.448.5159 (fax)
212.448.5190 or 5110 (lab)





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