rozenkov at netscape.net
Sun Nov 20 20:47:54 EST 2005
If your colleague is concerned about possible effect of CD8 on subsequent tetramer binding, an option could be to negatively sort CD8 cells using a mixture of antibodies CD4, CD19, CD13, etc. Another, electronical option of CD8 sorting that can reduce listmode file size and facilitate file manipulation and analysis is "live gating" when only events from a designated gate (e.g., CD8+) are saved into a lmd file.
In theory, CD8 can sterically interfere with tetramer binding, which may be different for different CD8 clones depending on the binding sites, and tetramer manufacturers recommend specific clones that can be used with tetramers. These CD8 antibodies are recommended for simultaneous staining with tetramers, so there should not be a problem with CD8 staining before sorting (in theory).
However, in our experience, we had difficulties with co-staining with tetramers and CD8, and we could demonstrate that using tetramer manufacturer's recommended and supplied CD8 and their standard procedure, CD8 antibodies reduced tetramer staining intensity (MFC) of tetramer positive cells and control T cell lines 2-5 times, so making it undistinguishable.
I think it would be interesting and of use to many of us to hear from other flow colleagues of their experience and solutions regarding tetramer-CD8 staining.
Vladislav Rozenkov, M.D., Ph.D.
Centre for Immune and Targeted Therapy
Division of Medicine
University of Queensland
rozenkov at netscape.net
From: Joanne Lannigan <joannelannigan at virginia.edu>
Sent: Mon, 7 Nov 2005 10:39:51 -0500
Subject: Tetramer binding and pre-selection of CD8 population
I am posting this question below for a colleague, I will be happy to forward responses. Thanks in advance-
Our lab is interested in analyzing the phenotypes (naïve, memory, effector) of human CD8-positive T cells that bind to peptides derived from self-proteins. In order to focus on this population, we thought that pre-selection of CD8-positive cells would facilitate our analysis. My question is would selection (positive or negative) of CD8 positive lymphocytes affect either the subsequent binding of tetramer or the phenotype of the cells that do bind, or potentially bias the results? We have seen in the literature that most studies are done on un-manipulated cell populations. The rarity of the cells in which we are interested makes the acquisition and analysis of bulk PBMC suspensions cumbersome and time consuming. Thank you.
Joanne Lannigan, MS
Director, Flow Cytometry Core Facility
University of Virginia
Jordan Hall, Room 7067
P.O. Box 800734
Charlottesville, VA 22908-0734
email: joannelannigan at virginia.edu
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