Gill Webster g.webster at genesis.co.nz
Wed Jul 18 15:32:14 EST 2001

The first thing that springs to mind is that 7AAD intermediate staining
could be revealing cells that are undergoing apoptosis.  This does not seem
an unlikely scenario for bone marrow cells, especially since it was fresh
material.  Was this sample froma healthy individual? Apoptotis in the bone
marrow could be a consequence of increased haematopoiesis or a marker of
disease (or the sample was handled badly!).  Most cell-impermeant viability
dyes, such as Propidium Iodide and Sytox Green, will reveal a dim positive
population if cells are stained for long enough.  This usually a consequence
of cell membranes becoming more permeable as a result of ongoing apoptosis.
This technique is not full proof, but can be a good indicator.  Check the
light scatter properties of the dim positive cells and compare that with the
negative and the bright positive.  Apoptotic lymphocytes do exhibit altered
light scatter characteristics compared to viable and necrotic cells.
Remember staining concentration is also a factor that will affect the
ability to detect the 7AAD dim + cells. You could confirm this staining
pattern on other lymphocyte cell lines induced to undergo apoptosis.  After
that, there are now many ways to measure apoptotis by flow

Gill Webster PhD
Head of Flow Cytometry
Genesis Research and Development
PO Box 50
New Zealand

email: g.webster at genesis.co.nz
direct dial 00 61 9 3743708
fax: 00 61 9 373 2189

-----Original Message-----
From: Jeff Louie [mailto:jlouie at hsc.usc.edu]
Sent: Wednesday, July 18, 2001 6:30 AM
To: cyto-inbox
Subject: 7 AAD

I have tried using 7AAD for viability using the procedure from Current
Protocols in Cytometry section 9.2.3. I made the stock solution using
the procedure found on 7.11.4.  I am using a FacsCalibur flow routinely
used for three color applications and selected the Percp setting for my
analysis. The results on one bone marrow sample demostrated clear
negative and positive populations, but included a moderately bright 7AAD
positive population between the neg and pos population. The positive
population was determined/verified by setting up a previously fixed
sample, CDChex,  as a positive control and the bone marrow sample used
was tested on the same day it was collected. I would like to have some
imput and a few alternate procedures to follow to eliminate the
indeterminate populations or better understand the significance of the
moderately bright 7AAD population on a fresh sample.


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