Macropinosome quantitation assay

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.

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