Analysis and isolation of endocytic vesicles by flow cytometry and sorting: demonstration of three kinetically distinct compartments involved in fluid-phase endocytosis.

The existence of three distinct classes of endocytic vesicles that are part of the pathway of fluid-phase endocytosis has been demonstrated by flow cytometry. Amounts of fluorescent and scattered light were measured on a particle-by-particle basis for unfractionated whole cell lysates from cells incubated with fluorescein isothiocyanate-dextran. After 20 min two different fluorescent populations were observed, and after a 180-min incubation a third highly fluorescent population was found. Since the fluorescein isothiocyanate-dextran per fluid-phase vesicle should be a function solely of the external fluorescein isothiocyanate-dextran concentration, the existence of endocytic compartments with widely different amounts of fluorescence could result from a wide range of sizes of initial endocytic vesicles. However, the kinetics of appearance of the intermediate and highly fluorescent vesicles suggest that these compartments become labeled through fusion with the smaller primary endocytic vesicles.