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Automated Imaging and Analysis for the Quantification of Fluorescently Labeled Macropinosomes
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Macropinosome quantitation assay.

Jack T H Wang1, Rohan D Teasdale2, David Liebl3

  • 1Institute 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.

Methodsx
|July 8, 2015
PubMed
Summary
This summary is machine-generated.

This study presents a refined assay for quantifying macropinocytosis, a cellular process involving fluid uptake. The method accurately measures the rate and volume of fluid-phase endocytosis in adherent cells.

Keywords:
AmilorideDextranFluid-phase endocytosisFluorescence microscopyMacropinocytosisMacropinosomeQuantitation of macropinocytosis

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Area of Science:

  • Cell Biology
  • Molecular Biology
  • Biochemistry

Background:

  • Macropinocytosis is a form of endocytosis distinct from phagocytosis, driven by actin cytoskeleton remodeling and growth factor receptor signaling.
  • Previous methods for assessing macropinocytosis, such as that by Wang et al. (2010), allowed for quantitative assessment of macropinosome biogenesis and maturation.
  • Perturbing protein function via pharmacological inhibitors or genetic manipulation (knock-down/knock-out) is crucial for studying macropinocytosis.

Purpose of the Study:

  • To describe a modified quantitative protocol for measuring fluid-phase uptake in adherent cells.
  • To enable precise measurement of the rate and volume of macropinosomes.
  • To provide a customizable assay applicable to various cell types and experimental conditions.

Main Methods:

  • Utilizes fluorescent dextran as a fluid-phase marker.
  • Employs microscopy for imaging.
  • Incorporates semi-automated image analysis for quantitation of macropinosomes in individual cells.

Main Results:

  • The modified protocol allows for the quantitation of macropinosomes within a large number of individual cells.
  • The assay is effective even in non-homogenous cell populations, including transiently transfected cell monolayers.
  • The method provides quantitative data on the rate and volume of fluid uptake.

Conclusions:

  • The described protocol offers a reliable and adaptable method for quantifying macropinocytosis.
  • This assay facilitates detailed studies into the mechanisms and regulation of macropinocytosis.
  • The protocol's flexibility supports its application in diverse cell biology research settings.