Hi everybody, I am puzzled by a question that I hope someone can help me understand. We are using a human lymphocyte cell-surface protein extracellular domain that, through stable transfection, is being produced in insect cells. Although this antigen itself is well-characterized, and it seems to be a receptor protein, no counter-ligand has been definitely found yet. Here is the problem. We have biotinylated the protein and are testing a wide range of different cell types for the ability to specifically bind it. When we test unfixed cells by flow cytometry, we either get no staining (using either avidin-FITC or -PE) or when there seems to be some staining, it is merely 1-10% of the primary or cell line cells with a large range of intensities (starting at barely above the control-determined +/- level). So far so good, however, when a guy here did side-by-side fluorescence confocal microscopy, he used cells that either were or were not formaldehye-fixed prior to treatment with protein followed by the avidin-fluorochrome; the fixed cells of a particular type showed a definitely positive fraction of ~50%, while the unfixed cells had a positive percentage similar to the unfixed cells examined by FCM. When previously fixed cells were tested using FCM, a tightly-focussed, reasonably intense positive population of ~60% appeared, whereas in the unfixed sample the positivity was only 5% with the usual scattered intensity. The staining can be partially blocked using unbiotinylated ligand. I don't think the staining of the fixed cells is nonspecific because cell types that were totally negative when unfixed remain negative when fixed. It seems contrary to sense that if a cell has a specific receptor for a ligand, it only becomes highly evident using fixed cells and is questionable when using healthy, unfixed cells. Does anyone have experience with this phenomenon? What does it mean? Kevin G. Waddick, Ph.D. Staff Scientist Hughes Institute 2685 Patton Road Roseville, Minnesota 55113 (612) 628-0598 (612) 628-9891 Fax
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