Dear Cindy, there were some reports on receptor capping done in flow cytometer. During cap formation on one pole of the cell (which is not 100% true in all cases; sometimes you could see only patches all over the cell) you should be able to see slightly higher height of the pulse, but more importantly much lower width of the pulse (if your cytometer supports pulse processing). Also the laser beam width could be optimized for the particular cell size. The cap should be rather small to minimize the signal dependence on the cap orientation towards the laser beam. I have tried that several years ago to detect and quantify cocapping of different molecules with reproducible results which, unfortunately, have not been published. In my setup the cells were prelabeled with different fluorescent antibodies (Fab fragments) together with unlabeled triggering antibody (whole IgG), washed and then the capping was induced using Fc part specific polyclonal. You should play a little bit with timing and temperature to induce really nice caps. If you see just patches all over the cell surface under the microscope, it it not worth to try flow cytometry. If you are interested I will try to find the relevant references. Karel Drbal Institute for Immunology Vienna International Research Cooperation Center University of Vienna "Bristow, Cindy" wrote: > > Do you think fluorescence intensity of a receptor should increase or > decrease by flow as the result of receptor capping? In addition to steric > hindrance of antibody binding to capped receptors, would capping interfere > with detection as receptors whirl through the flow chamber? Any thoughts? > Much thanks. > > Cynthia L. Bristow, PhD > University of North Carolina Hospitals > cbristow@unch.unc.edu <mailto:cbristow@unch.unc.edu>
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