Dear David,
upon stimulation the CD4+ T lymphocyte will 'loose' its CD4. It's
internalized during this stimulation process. Even taking that in account it
can also be that:
1. you picked that one CD4 antibody that doesn't recognize the fixed
molecule anymore.
2. you stain your cells before fixation and permeabilization, which is due
to a lower CD4 detection (internalization). Using the CD4 antibody together
with your anti-cytokine antibodies might already give a better result.
I remember that there was this question before and several of us gave
suggestions. Try the archive:
http://www.cyto.purdue.edu/hmarchiv/index.htm
Although downregulated I often can detect the CD4. Even with a PerCP labeled
antibody (BD).
Joost
____________________________________________________
J.H.N.Schuitemaker
Laboratory Manager / Senior Research Technician
Cellular Immunology Group
Cell Biology & Histology
Academic Medical Center
P.O.box 22700
1100 DE Amsterdam
The Netherlands
Telephone +31 (0)20 5664960
FAX +31 (0)20 6974156
Internet: http://www.amc.nl
http://home.wanadoo.nl/flowcytometry
-----Original Message-----
From: David Ritchie [mailto:dritchie@malaghan.org.nz]
Sent: woensdag 16 oktober 2002 20:48
To: cyto-inbox
Subject: CD4 on stimulated human T cells
Any suggestions welcomed on below problem,
We are tracking the cytokine profiles (by intracellular staining) produced
by CD4+ T cells in patients who have undergone allogeneic bone marrow
transplant (BMT). We are looking at cytokine production pre- and
post-stimulation of T cells with PHA/ionomycin for 4 hours. We have no
difficulty clearly identifying CD4+ T cells on the unstimulated sample, but
on the stimulated sample CD4 seems to disappear. CD3 is still clearly
evident. Has anyone seen this before? Is the cell fixing process used for ic
staining likely to interfere with CD4 expression or anti-CD4 binding? We
don't seem to be losing cells in the short culture period.
Thanks for any comments
David Ritchie
This archive was generated by hypermail 2b29 : Sun Jan 05 2003 - 19:26:27 EST