Visualizing the Early Stages of Phagocytosis

Abstract

The mammalian body is equipped with various layers of mechanisms that help to defend itself from pathogen invasions. Professional phagocytes of the immune system - such as neutrophils, dendritic cells, and macrophages - retain the innate ability to detect and clear such invading pathogens through phagocytosis. Phagocytosis involves choreographed events of membrane reorganization and actin remodeling at the cell surface. Phagocytes successfully internalize and eradicate foreign molecules only when all stages of phagocytosis are fulfilled. These steps include recognition and binding of the pathogen by pattern recognition receptors (PRRs) residing at the cell surface, formation of phagocytic cup through actin-enriched membranous protrusions (pseudopods) to surround the particulate, and scission of the phagosome followed by phagolysosome maturation that results in the killing of the pathogen. Imaging and quantification of various stages of phagocytosis is instrumental for elucidating the molecular mechanisms of this cellular process. The present manuscript reports methods to study the different phases of phagocytosis. We describe a microscope-based approach to visualize and quantify the binding, phagocytic cup formation, and the internalization of particulate by phagocytes. As phagocytosis occurs when innate receptors on phagocytic cells encounter ligands on a target particle bigger than 0.5 µm, the assays we present here comprise the use of pathogenic fungi Candida albicans and other particulates such as zymosan and IgG-coated beads.

Sphingomyelin Biosynthesis Is Essential for Phagocytic Signaling during Mycobacterium tuberculosis Host Cell Entry

“…Phagocytosis assay. The phagocytosis assay was performed as previously described (62). Briefly, fixed cells were washed twice with PBS and incubated for 15 min in permeabilization buffer (0.1% Triton X-100 and 1% bovine serum albumin [BSA]).…”

Research progress on the immune mechanism of the silkworm Bombyx mori

“…In addition to the immune mechanisms described above, phagocytosis, a evolutionarily conservative immune mechanism, commonly exists in invertebrates and can remove the invasive pathogens and apoptotic residues (Lü, 2008;Ye et al, 2012;Chen et al, 2015). This process is accomplished in a single cell, involving the identification, phagocytosis, destruction of invasive pathogens and death of cells themselves (Williams, 2007;Gray & Botelho, 2017;Rashidfarrokhi et al, 2017). Meanwhile, PGRPs, βGRPs, lysozyme, hemolin and lectins also play important roles in the innate immunity of the silkworm (Ochiai & Ashida, 1988Lee & Brey, 1995;Yoshida et al, 1996;Gandhe et al, 2007).…”