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Wang JT, Teasdale RD, Liebl D. Macropinosome quantitation assay. MethodsX. 2014;1:36-41doi: 10.1016/j.mex.2014.05.002.
Wang, J. T., Teasdale, R. D., & Liebl, D. (2014). Macropinosome quantitation assay. MethodsX, 136-41. https://doi.org/10.1016/j.mex.2014.05.002
Wang, Jack T H, et al. "Macropinosome quantitation assay." MethodsX vol. 1 (2014): 36-41. doi: https://doi.org/10.1016/j.mex.2014.05.002
Wang JT, Teasdale RD, Liebl D. Macropinosome quantitation assay. MethodsX. 2014 Jun 02;1:36-41. doi: 10.1016/j.mex.2014.05.002. eCollection 2014. PMID: 26150932; PMCID: PMC4472846.
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MethodsX. 2014 Jun 02;1:36-41. doi: 10.1016/j.mex.2014.05.002. eCollection 2014.

Macropinosome quantitation assay.

MethodsX

Jack T H Wang, Rohan D Teasdale, David Liebl

Affiliations

  1. 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.
  2. Institute for Molecular Bioscience, The University of Queensland, St. Lucia, Brisbane 4072, Queensland, Australia.
  3. 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.

PMID: 26150932 PMCID: PMC4472846 DOI: 10.1016/j.mex.2014.05.002

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

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