Flow cytometry distinction between adherent and phagocytized yeast particles

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Abstract
Our laboratory recently developed a light microscopy staining technique that provides a mean to distinguish between yeast that are simply bound to the surface of macrophages and yeast that have actually been phagocytized by macrophages (7). We adapted this technique by using fluorescent probes in order to test phagocytic activity by flow cytometry. Thus we are able to distinguish unambiguously extracellular from intracellular yeast during phagocytosis with the fast rate of flow cytometry (∼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 external pH equilibrated by 50 μ monensin 30 min/4°C) protected from the action of tannic acid, a nonmembrane permeable molecule. The results presented here validate this new technique. An application is presented showing the inhibition of endocytosis by cytochalasin‐B.