Folks:
This message is in reply to Dr. Ashraf Abdel
Hafez's question regarding use of Azide. I answer
this (below) and am also posing my own question
regarding application of intracellular staining for
cytokines in PBMC.
AZIDE/ANSWER:
Dr. Hafez:
Salaam Aliekum! My understanding is that beyond
antimicrobial activity, azide shuts down transmembrane
trafficking (which may continue even in fairly harsh
conditions following isolation of cells or termination
of cultures). If this is not done, significant
amounts of fluorophore-bound Ab may be internalized -
resulting in signal degradation. Since staining
times are short, and the fluorophore in any case stays
with the cell, this is a minor problem, but it can
complicate cluster analysis once the samples are
processed.
I have stained with and without azide and have
seen no major differences. However it's probably
better to use it if you have the opportunity.
KUWAIT PROBLEM/QUESTION:
My colleagues and I in Kuwait would like to
identify a facility (preferably in the USA or Europe
making use of intracellular cytokine analysis of PBMC
in patient teatment. The method is used routinely
in research, but I'm not aware of any medical centers
using it clinically.
Our need to establish liason with such a facility
and adapt this approach to patient care in Kuwait is
immediate. Since the country appears to be a
primary staging area for the US invasion of Iraq, we
assume there will be retaliatory strikes against us.
Since it is also a safe bet that this will expose a
significant fraction of our population to potentially
immunomodulatory toxins of various types, FACS based
screening has become a priority.
Our own group, for better or worse is on the
cutting edge of this effort. For the past decade we
have been investigating the underlying immunological
mechanisms of disease increases among the civil
population of Kuwait which occurred following the
(first!) Gulf War. At present, we would like to
provide Kuwaiti primary care facilities with
state-of-the-art capabilities for immunological
analysis of large numbers of people.
I would be grateful if replies to this were
addressed to our principal investigator in Kuwait:
Fadia F. Mahmoud, Ph.D.
Department of Medical Laboratory Technology
Kuwait University Faculty of Health Sciences and
Nursing
31470 Suliebikhat, Code 90805
State of Kuwait
TEL: KUWAIT 011 965 482-4769
FAX: 011 965 483-3631
email: fadia@hsc.kuniv.edu.kw
fadia101@yahoo.com
Thank You
Sincerely
David D. Haines, Ph.D.
Dept. Medicine,
University of Connecticut Health Center
MESSAGE FROM DR. ABDELHAFEZ:
Date: Wed, 25 Sep 2002 05:28:58 -0700 (PDT)
From: "Dr. Ashraf Abdelhafez"
<asabdelhafez@yahoo.com>
Subject: Azide?
To: "Cytometry Mailing List"
<cytometry@flowcyt.cyto.purdue.edu>
Hi All:
Does anybody have a conclusive answer to why azide is
added to staining buffer. I have found information
that says that it functions to inhibit microbial
growth. I have found that it can be used to stop
reactions. I have found people recommending not to use
in certain experiment because it can alter protein
function. And I have found people saying that you do
not need to put if you are going to analyze the cells
within hours. But I was not able to find information
on that exact reason for adding it to staining buffer.
I would like to hear what you have. Only what you
believe is a conclusive answer is accepted.
Thanks,
Ashraf
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