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Methods for detecting internalized, FM 1-43 stained particles in epithelial cells and monolayers.

C A Bertrand1, C Laboisse, U Hopfer

  • 1Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA. cbertra@pitt.edu

Biophysical Journal
|August 29, 2006
PubMed
Summary

This study introduces a new method to quantify membrane trafficking in epithelial cells using FM 1-43 dye. The technique measures retained fluorescent particles after dye washout, offering a reliable alternative for exocytosis studies.

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Area of Science:

  • Cell Biology
  • Membrane Trafficking
  • Epithelial Physiology

Background:

  • The membrane dye FM 1-43 is commonly used for exocytosis quantification in neurons.
  • In epithelial cells, FM 1-43 often yields inconsistent results due to intracellular staining and baseline fluctuations.
  • A novel approach is needed to accurately measure membrane trafficking in epithelial systems.

Purpose of the Study:

  • To develop and validate a new method for quantifying membrane trafficking in epithelial cells.
  • To assess the reliability of using retained fluorescent puncta after FM 1-43 washout as a measure of exocytosis.
  • To compare this method across different epithelial cell preparations (clusters vs. monolayers).

Main Methods:

  • Quantified retained fluorescent puncta after FM 1-43 dye washout.
  • Utilized serial sectioning with epifluorescence or confocal microscopy for image acquisition.
  • Applied the method to intestinal goblet cell line clusters and filter-grown monolayers.
  • Stimulated exocytosis using ATP and compared results with capacitance measurements.

Main Results:

  • The algorithm consistently identified internalized particles regardless of microscopy type (epifluorescence vs. confocal).
  • Low-density cell clusters showed significantly more internalized particles than monolayers or high-density clusters.
  • ATP stimulation markedly increased internalized particle counts across all preparations.
  • Quantified particle internalization correlated well with capacitance measurements of exocytosis.

Conclusions:

  • Retained fluorescent puncta after FM 1-43 washout provide a robust method for quantifying membrane trafficking in epithelial cells.
  • This technique is effective for both unpolarized cells and intact monolayers.
  • The method offers a single, reliable approach to study exocytosis and endocytosis in diverse epithelial models.