title: Macropinosome quantitation assay.
abstract: In contrast to phagocytosis, macropinocytosis is not directly initiated by interactions between cell surface receptors and cargo ligands, but is a result of constitutive membrane ruffling driven by dynamic remodelling of cortical actin cytoskeleton in response to stimulation of growth factor receptors. Wang et al. (2010) [13] developed a reliable assay that allows quantitative assessment of the efficiency and kinetics of macropinosome biogenesis and/or maturation in cells where the function of a targeted protein has been perturbed by pharmacological inhibitors or by knock-down or knock-out approaches. In this manuscript we describe a modified quantitative protocol to measure the rate and volume of fluid phase uptake in adherent cells. This assay:•uses fluorescent dextran, microscopy and semi-automated image analysis;•allows quantitation of macropinosomes within large numbers of individual cells;•can be applied also to non-homogenous cell populations including transiently transfected cell monolayers. We present the background necessary to consider when customising this protocol for application to new cell types or experimental variations.
authors.affiliation: Institute for Molecular Bioscience, The University of Queensland, St. Lucia, Brisbane 4072, Queensland, Australia ; School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane 4072, Australia.
authors.forename: Jack T H
authors.initials: JT
authors.lastname: Wang
authors.affiliation: Institute for Molecular Bioscience, The University of Queensland, St. Lucia, Brisbane 4072, Queensland, Australia.
authors.forename: Rohan D
authors.initials: RD
authors.lastname: Teasdale
authors.affiliation: Institute for Molecular Bioscience, The University of Queensland, St. Lucia, Brisbane 4072, Queensland, Australia ; Dept. Bacterial Pathogenesis and Cellular Responses, Institute of Sciences Research and Technologies/CEA-Grenoble, Grenoble, France.
authors.forename: David
authors.initials: D
authors.lastname: Liebl
keywords: Amiloride; Dextran; Fluid-phase endocytosis; Fluorescence microscopy; Macropinocytosis; Macropinosome; Quantitation of macropinocytosis