title: High-throughput quantification of early stages of phagocytosis.
abstract: Phagocytosis--the engulfment of cells and foreign bodies--is an important cellular process in innate immunity, development, and disease. Quantification of various stages of phagocytosis, especially in a rapid screening fashion, is an invaluable tool for elucidating protein function during this process. However, current methods for assessing phagocytosis are largely limited to flow cytometry and manual image-based assays, providing limited information. Here, we present an image-based, semi-automated phagocytosis assay to rapidly quantitate three distinct stages during the early engulfment of opsonized beads. Captured images are analyzed using the image-processing software ImageJ and quantified using a macro. Modifications to this method allowed quantification of phagocytosis only in fluorescently labeled transfected cells. Additionally, the time course of bead internalization could be measured using this approach. The assay could discriminate perturbations to stages of phagocytosis induced by known pharmacological inhibitors of filamentous actin and phosphoinositol-3-kinase. Our methodology offers the ability to automatically categorize large amounts of image data into the three early stages of phagocytosis within minutes, clearly demonstrating its potential value in investigating aberrant phagocytosis when manipulating proteins of interest in drug screens and disease.
authors.affiliation: Institute for Molecular Bioscience and NHMRC Program in Cellular Microbiology, The University of Queensland, Brisbane, Australia.
authors.forename: Jeremy Changyu
authors.initials: JC
authors.lastname: Yeo
authors.forename: Adam Alexander
authors.initials: AA
authors.lastname: Wall
authors.forename: Jennifer Lea
authors.initials: JL
authors.lastname: Stow
authors.forename: Nicholas Ahti
authors.initials: NA
authors.lastname: Hamilton
mesh_terms: D000818:Animals; D002232:Carbocyanines / Q000032:analysis / Q000378:metabolism; D002460:Cell Line; D002867:Chromones / Q000494:pharmacology; D015638:Cytochalasin D / Q000494:pharmacology; D003584:Cytological Techniques / Q000379:methods*; D004912:Erythrocytes / Q000737:chemistry / Q000378:metabolism; D005456:Fluorescent Dyes / Q000032:analysis / Q000378:metabolism; D057166:High-Throughput Screening Assays / Q000379:methods; D007091:Image Processing, Computer-Assisted / Q000379:methods*; D007074:Immunoglobulin G / Q000378:metabolism; D051379:Mice; D008856:Microscopy, Fluorescence / Q000379:methods*; D008863:Microspheres; D009025:Morpholines / Q000494:pharmacology; D010587:Phagocytosis / Q000187:drug effects / Q000502:physiology*; D015203:Reproducibility of Results; D012756:Sheep
chemical_list: D002232:Carbocyanines; D002867:Chromones; D005456:Fluorescent Dyes; D007074:Immunoglobulin G; D009025:Morpholines; C089187:cyanine dye 3; D015638:Cytochalasin D; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one
keywords: ImageJ; assay; high throughput; image-based; macrophage; phagocytosis