FLOW-CYTOMETRY DISTINCTION BETWEEN ADHERENT AND PHAGOCYTIZED YEAST PARTICLES

Our laboratory recently developed a light microscopy staining techniqu e that provides a mean to distinguish between yeast that are simply bo und to the surface of macrophages and yeast that have actually been ph agocytized by macrophages (7). We adapted this technique by using fluo rescent probes in order to test phagocytic activity by flow cytometry. Thus we are able to distinguish unambiguously extracellular from intr acellular yeast during phagocytosis with the fast rate of flow cytomet ry (similar to 200 cells/s). The fluorescence quenching induced by a 1 % tannic acid solution (w/v) can be applied to any FITC-labeled, heat- killed yeast cell or bacteria. The yeast cells already engulfed in the macrophage remain with their native fluorescence (internal and extern al pH equilibrated by 50 mu M monensin 30 min/4 degrees C) protected f rom the action of tannic acid, a nonmembrane permeable molecule. The r esults presented here validate this new technique. An application is p resented showing the inhibition of endocytosis by cytochalasin-B. (C) 1994 Wiley-Liss, Inc.