Katie:
If what you want is the least disruptive method possible, maybe this is
an option:
1) pick a DNA specific, membrane PERMEABLE stain that suits your
cytometer and the next staining steps. Mol probes has as many options
as there are ice cream flavors (OK, *almost* as many). (no commercial
affiliation to mol probes, I just like them).
2) add PI or an alternative that suits your color config. Did I mention
mol probes has a lot of them?
3) add annexin V, also of the color that suits your config.
All of these have to be properly titrated, of course.
When acquiring the cells, trigger on fluorescence for the DNA dye in
step #1. That should get you rid of adult RBCs. Note that you'll still
have reticulocytes, which at ~1% of all RBCs would still outnumber your
WBCs, but now your scatter profile should be less hectic. Annexin V and
PI should give you then the info you want.
I don't know what cytometer you have, but if compensation is an issue
and you can't find a suitable annexin V conjugate, what I did is I
bought purified Annexin V and conjugated it myself. It was even cheaper
than buying most annexin V cojugates that are not fluorescein or PE.
On the option of annexin alternatives, I've seen some mol probes stuff
that shows DNA dyes that are less excluded than PI, and they showed
good correlation with annexin V status. From my experience with similar
dyes/situations, I am pretty sure it can work but expect to take your
time titrating the time, concentration and temperature needed to find
the spot where the correlation works best, and try to stick to it as
much as possible.
Your concern with annexin V and membrane fragments made me remember
something that I did some time ago. I was testing biostatus' cygel
(mounting material compatible with live cells) on a sample that I
stained with annexin V. Since they were llive cells I could use it. I
hurried a bit too much and when I put the slide on the microscope parts
of the cygel were still liquid. To my amazement I saw thousands of
little cell fragments floating and rushing in the "rivers" of cygel
that had formed between the solidified parts.I've got to make a video
of it sometime to show around, it is really a nice sight.
Good luck,
Uriel.
--
Uriel Trahtemberg, M.Sc.
MD/PhD student
The Laboratory for Cellular and Molecular Immunology
The Hebrew University - Hadassah Medical Organization
Jerusalem - ISRAEL
kfurr@bidmc.harvard.edu wrote:
Hi, Our group is
trying to develop an assay that will allow us to determine apoptotic
and necrotic lymphocytes (primarily apoptotic cells) in rhesus macaque
whole blood samples. In the future, we would like to avoid enrichment
of lymphocytes by density gradient separation since these procedures
can significantly change lymphocyte subset distributions. We are
currently using Annexin V and would like to continue to use Annexin V.
However, the problem we are running into is how to eliminate RBCs
without increasing the affinity for or losing Annexin V binding. Any
suggestions as to a procedure that would allow us to reduce RBCs
without affecting the integrity of the Annexin V binding or possibly an
alternative method to Annexin V staining that will not be affected by
the RBC lysis step would be greatly appreciated.
Many thanks in
advance,
Katie
Kathryn Furr
Division of Viral Pathogenesis
Beth Israel Deaconess Medical
Center
RE-111a
41 Avenue Louis
Pasteur
Boston, MA 02115
Phone :
617-667-4707
Fax :
617-667-8210
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Received on Tue Jan 29 14:38:00 2008
