QUANTITATIVE FLUORESCENCE ANALYSIS OF CYCLOSPORINE BINDING TO HUMAN LEUKOCYTES

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Abstract
The purpose of this investigation was to estimate the binding of cyclosporine at the single-cell level on human peripheral lymphocytes, and to test possible identity of the cyclosporine-binding site with a common receptor of T cell activation. A dansyl-coupled derivative (Dans cayclosprine) was used as a fluorescent probe. The histograms of unseparated, labeled peripheral leukocytes obtained by a fluorescence-activated cell sorter (FACS) showed that Dans cyclosporine stained all leukocytes-but two distinct populations could be separated based on the intensity of fluorescence. The more brightly labeled cells consisted mainly of granulocytes adn monocytes, whereas the less-bright cells represented the lymphocyte compartment. Fluorescence microscopy revealed binding on the membrane for both cell populations; the label was, however, rapidly internalized in phagocytes. For both populations binding was saturable, time and temperature dependent, and reversible. Half-saturation occurred at ∼ 5 × 10−7 M (kd). With respect ot lymphocyte subpopulations, no difference of cellular fluores-

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