Macropinosome quantitation assay

MethodsX. 2014 Jun 2;1:36-41. doi: 10.1016/j.mex.2014.05.002. eCollection 2014.

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.

Keywords: Amiloride; Dextran; Fluid-phase endocytosis; Fluorescence microscopy; Macropinocytosis; Macropinosome; Quantitation of macropinocytosis.