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Related Experiment Video

Updated: Jun 19, 2026

High-throughput Measurement of Plasma Membrane Resealing Efficiency in Mammalian Cells
10:07

High-throughput Measurement of Plasma Membrane Resealing Efficiency in Mammalian Cells

Published on: January 7, 2019

A plate reader-based method for cell water permeability measurement.

R A Fenton1, H B Moeller, S Nielsen

  • 1The Water and Salt Research Center, Institute of Anatomy, University of Aarhus, Aarhus C, Denmark.

American Journal of Physiology. Renal Physiology
|November 6, 2009
PubMed
Summary
This summary is machine-generated.

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This study introduces a novel, plate reader-based method for measuring cell volume and water permeability in mammalian cells. The new approach efficiently analyzes aquaporin-2 (AQP2) function and is suitable for screening AQP inhibitors.

Area of Science:

  • Cellular Physiology
  • Biophysics
  • Molecular Biology

Background:

  • Traditional cell volume and water permeability measurements are often time-consuming and unsuitable for confluent cell monolayers.
  • Existing methods limit the study of aquaporin (AQP) function in adherent cell lines.

Purpose of the Study:

  • To adapt a calcein-quenching assay for a plate reader to measure cell volume and water permeability in cultured mammalian cells.
  • To develop a method applicable to confluent epithelial cell monolayers and facilitate AQP research.
  • To investigate the role of aquaporin-2 (AQP2) phosphorylation in cell volume regulation.

Main Methods:

  • Developed a calcein-quenching assay adaptable to a plate reader for measuring cell volume changes.
  • Established a standard curve correlating fluorescence intensity with extracellular osmolyte concentrations (185–585 mosmol/kgH2O).

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Measuring the Osmotic Water Permeability Coefficient (Pf) of Spherical Cells: Isolated Plant Protoplasts as an Example
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  • Measured average cell volumes using a Coulter counter and derived an equation to model cell volume changes from fluorescence data.

    • Main Results:

    • Successfully adapted the calcein-quenching assay for high-throughput analysis using a plate reader.
    • Quantified water permeability in Madin-Darby canine kidney cells expressing AQP2 phosphorylation site mutants.
    • Determined the order of water permeability as AQP2-S256D > AQP2 wild-type > AQP2-S256A.

    Conclusions:

    • The developed plate reader method provides an efficient way to study cell volume changes in adherent cell lines.
    • This method is valuable for investigating aquaporin function, particularly AQP2 trafficking and regulation.
    • The assay is adaptable for screening aquaporin inhibitors within chemical compound libraries.