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

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.