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

¹ Univ of Pittsburgh School of Medicine
² U539 INSERM, Nantes, Fr
³ Case Western Reserve University
⁴ Rosalind Franklin University of Medicine and Science
⁵ University of Pittsburgh School of Medicine

^* To whom correspondence should be addressed. E-mail: cbertra{at}pitt.edu.

Submitted on April 11, 2006
Revised on May 8, 2006
Accepted on 31 July 2006

	Abstract

The membrane dye FM 1-43 has frequently been used to quantify exocytosis in neurons. In epithelia, intense lateral intracellular space staining and fluctuations in baseline labeling produced inconsistent results. Membrane retrieved in the presence of FM 1-43 retains the dye, however, and cells that undergo compensatory endocytosis during and following evoked exocytosis contain punctate, fluorescent particles following washout of external stain. As an alternative measure of trafficking, we quantified the fluorescent puncta retained after dye washout, and tested our method on both coverslip-grown cell clusters and filter-grown intact monolayers. Images for analysis were acquired using serial sectioning with either epifluorescence or confocal microscopy. Tests with an intestinal goblet cell line that exhibits basal and ATP-stimulated granule trafficking confirmed that: 1) the algorithm identified the same number of internalized particles with either epifluorescence or confocal microscopy acquired images; 2) low density clusters exhibited significantly more internalized particles per cell than either filter grown monolayers or high density clusters; 3) ATP stimulation significantly increased the number of internalized particles in all preparations; and 4) the number of particles internalized was comparable to capacitance measurements of exocytosis. This method provides a single technique for quantifying membrane trafficking in both monolayers and unpolarized cells.

Key Words: exocytosis, granules, membrane capacitance, mucin secretion