A fluorescent microplate assay for exocytosis in alveolar type II cells

A Wemhöner, Manfred Frick, Paul Dietl, P Jennings, T. Haller

Research output: Contribution to JournalArticleAcademicpeer-review

Abstract

The authors describe a simple, reliable, and quantitative assay to monitor exocytotic fusion of lamellar bodies (LBs) in adherent rat alveolar type II (AT II) cells. The assay is based on fluorescence measurements of LB-plasma membrane (PM) fusions modified for the use in multiwell culture plates to obtain a high-sample throughput. In particular, it is based on the presence of a highly light-absorbing dye in the cell supernatants to increase the specificity of fluorescence signals and to yield pseudo-confocal information from the cells. When the assay was tested with agonist-(ATP) and phorbolester-induced stimulation of LB-PM fusions, the authors found a good correlation with direct microscopic investigations based on single cell recordings. To further validate the assay, they used Curosurf at 10 mg/ml. However, it influenced neither the basal nor the ATP-stimulated rate of LB-PM fusions. This was corroborated by the fact that Curosurf had no effect on resting Ca (2+) levels nor the ATP induced Ca (2+) signals. The results cast new light on previous findings that surfactant phospholipids decrease the rate of secretion in AT II cells in a dose-dependent way. The authors conclude that the inhibitory effect exerted by phospholipids might be due to action on a later step in exocytosis, probably associated with exocytotic fusion pore expansion and content release out of fused vesicles.

Original languageEnglish
Pages (from-to)286-95
Number of pages10
JournalJournal of Biomolecular Screening
Volume11
Issue number3
DOIs
Publication statusPublished - Apr 2006

Keywords

  • Adenosine Triphosphate
  • Animals
  • Exocytosis
  • Fluorescence
  • Male
  • Pulmonary Alveoli
  • Rats
  • Rats, Sprague-Dawley
  • Tetradecanoylphorbol Acetate
  • Journal Article
  • Research Support, Non-U.S. Gov't

Fingerprint Dive into the research topics of 'A fluorescent microplate assay for exocytosis in alveolar type II cells'. Together they form a unique fingerprint.

Cite this