Flow analysis of MHC molecules and other membrane proteins in isolated phagosomes

Abstract

A method was developed to apply flow cytometry analysis to the characterization of individual phagosomes. Macrophages were incubated with latex 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 properties of latex bead phagosomes allowed their analytic isolation from other organelles and cell fragments by flow analysis using a narrow gate based on scatter parameters. Furthermore, separate gates were established for phagosomes containing one, two and even three beads, which were sorted and examined by electron microscopy (EM). EM showed that the phagosomal membrane was closely apposed to the latex bead in most phagosomes, but some more spacious phagosomes were also observed. Phagosomes were immunolabeled and subjected to flow analysis for MHC-I and MHC-II molecules and lysosomal membrane markers (LAMPs). The proportion of LAMP-positive phagosomes increased with incubation time, reflecting maturation of phagolysosomes. Significant staining for MHC-I and MHC-II was demonstrated and remained relatively constant with time. Flow analysis of phagosomes allows the characterization and comparison of individual phagosomes, and the identification of subpopulations of phagosomes with differing membrane compositions. It also provides the advantage 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 composition as a function of phagosome maturation.