FLOW-ANALYSIS OF MHC MOLECULES AND OTHER MEMBRANE-PROTEINS IN ISOLATED PHAGOSOMES

A method was developed to apply flow cytometry analysis to the charact erization of individual phagosomes. Macrophages were incubated with la tex beads and homogenized to release the phagosomes. Intact cells and nuclei were removed by low speed centrifugation, and a crude phagosome preparation was fixed with paraformaldehyde. Distinct optical propert ies of latex bead phagosomes allowed their analytic isolation from oth er organelles and cell fragments by flow analysis using a narrow gate based on scatter parameters. Furthermore, separate gates were establis hed for phagosomes containing one, two and even three beads, which wer e sorted and examined by electron microscopy (EM). EM showed that the phagosomal membrane was closely apposed to the latex bead in most phag osomes, but some more spacious phagosomes were also observed. Phagosom es were immunolabeled and subjected to flow analysis for MHC-I and MHC -II molecules and lysosomal membrane markers (LAMPs). The proportion o f LAMP-positive phagosomes increased with incubation time, reflecting maturation of phagolysosomes. Significant staining for MHC-I and MHC-I I was demonstrated and remained relatively constant with time. Flow an alysis of phagosomes allows the characterization and comparison of ind ividual phagosomes, and the identification of subpopulations of phagos omes with differing membrane compositions. It also provides the advant age of analytically isolating phagosomes from other components of the cell without the need for extensive prior physical purification. Thus, it can be used to rapidly assess changes in phagosomal membrane compo sition as a function of phagosome maturation. (C) 1998 Elsevier Scienc e B.V. All rights reserved.