[Cytometry] removing azide from antibodies
Peter.Lopez at med.nyu.edu
Wed May 2 09:56:02 EDT 2012
Agreed. This is our experience with calcium mobilization studies in flow, where it's best to use non-azide containing antibodies for real-time activation studies. Otherwise we have never seen a cell growth issue after sorting that we attributed to the use of azide-containing antibodies. Having azide in your wash buffer is a different story, but I don't think people do that too much anymore.
Research Assistant Professor of Pathology
Director, Core Cytometry Facility, Office of Collaborative Science
NYU School of Medicine
540 First Ave Skirball 2nd floor Administration
New York, NY 10016
Peter.Lopez at med.nyu.edu
From: cytometry-bounces at lists.purdue.edu [mailto:cytometry-bounces at lists.purdue.edu] On Behalf Of John Seavitt
Sent: Tuesday, May 01, 2012 12:59 PM
To: Cytometry Mailing List
Subject: Re: [Cytometry] removing azide from antibodies
I am wondering if perhaps the researcher is over-extrapolating from experiments wherein antibody is added directly to cells in culture (i.e., crosslinking of lymphocyte antigen receptors and co-stimulation receptors). BD certainly sells azide-free antibodies for this latter purpose, and it may be that the azide could have an effect in that setting. Regardless, we've routinely stained, depleted, re-stained, sorted and then performed long term cell culture with azide-preserved antibodies without any issue.
John R. Seavitt, Ph.D.
Cutaneous Biology Research Center
Harvard Medical School
13th Street Boston, MA 02129
Phone: (617) 724-8280
FAX: (617) 726-4453
> -----Original Message-----
> From: cytometry-bounces at lists.purdue.edu
> [mailto:cytometry-bounces at lists.purdue.edu] On Behalf Of Guzik, Lynda
> Sent: 01 May 2012 16:43
> To: 'Cytometry Mailing List'
> Subject: [Cytometry] removing azide from antibodies
> Hi All,
> I had an interesting discussion for a researcher yesterday and thought
> I'd ask for a consensus of opinion from this learned forum.
> The researcher insists that he must remove the azide from his
> commercial antibody preps for sorting applications in order to ensure
> cell growth.
> The cells in question are mouse wbc's. He is using a size exclusion
> column to remove the azide, but has no way of monitoring if or how
> much of the azide has been removed. His antibodies are directly
> conjugated to a variety of fluorophores, none of which are tandem
> dyes. My feeling is that this is 'extra' work which will most likely
> cause more problems than it solves. The azide (at 0.02%
> concentration) should be diluted out enough during the staining and
> washing process as to have a negligible effect. None of my other
> researchers have had a problem with cell growth either in culture or
> in animal transplantation. And why introduce unknown variables into
> your experiment such as changes in concentration and P:F ratio -
> especially if these 'modified'
> are not being used in the day-to-day analysis panels. Purchase of
> de-free antibodies has been considered and rejected. What is the
> general feeling about this situation? Do you or any of your
> researchers routinely remove azide from their commercial antibodies?
> How do you verify that you have a) removed the azide and b) not harmed
> your conjugated antibody?
> Thank you-
> Lynda Guzik
> McGowan Institute
> Bridgeside Point II - Rm334
> 450 Technology Dr
> Pittsburgh, PA 15219
> guzilj at upmc.edu<mailto:lguzilj at upmc.edu>
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