Keith asks about Ag downregulation after PBMC stimulation.... 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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