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Yes, these antigens are downregulated! In the case of CD4, it is internalized
via endocytosis through a PKC-mediated phosphorylation signal. This
internalization is probably the most common mechanism for downregulation.
Another antigen, CD62L, is proteolytically removed from the cell surface by a
metalloproteinase.
We have not found drugs which inhibit the internalization (without severely
screwing up the cells). However, we have figured out how to get around it...
it's rather simple. Just pre-stain the cells with the monoclonals to antigens
which are internalized. Before we stimulate PBMC, we "stain" the cells at room
temperature (NO AZIDE!) with the conjugated CD4 (the cells are concentrated to
be able to stain in a 50 ul volume). The cells are washed with medium, and
resuspended at 37C in stimulation medium. They are then stimulated.
There is still a small problem: the endosome is acidic, reducing FITC
fluorescence, and it is proteolytic, reducing phycobiliprotein (PE)
fluorescence. After 6 hours, this reduces the fluorescence of either
fluorochrome about 10x. This still allows for adequate resolution of CD4 T
cells, however. We have found that if you use Monensin to force accumulation in
the Golgi (instead of the considerably more expensive Brefeldin A), then you
have the side benefit that endosomal pH is kept high and neither the FITC nor PE
conjugates are significantly affected.
Voila! Easy, positive ID of CD4 T cells in stimulated cultures! This method
will be published soon; look for an article by D. Mitra with myself as last
author.
mr
PS - CD62L requires different tricks; if you are interested, contact me
directly.
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CD-ROM Vol 3 was produced by Monica M. Shively and other staff at the
Purdue University Cytometry Laboratories and distributed free of charge
as an educational service to the cytometry community.
If you have any comments please direct them to
Dr. J. Paul Robinson, Professor & Director,
PUCL, Purdue University, West Lafayette, IN 47907.
Phone: (765)-494-0757;
FAX(765) 494-0517;
Web