RESUMO
BACKGROUND: Fluorescence membrane markers are efficient tools for visualizing the dynamics of membrane recycling processes in living cells. The outer hair cell (OHC) - a bipolar epithelial cell in the cochlea - possesses endocytic activity at both its apical and basal poles. The best visual overview of transcytosis in the OHC is achieved when the cell is isolated, so that both the apical and the basal poles are in the same focal plane to allow confocal imaging. Until now, fluorescent markers were applied to the extracellular environment of isolated OHCs without distinguishing the apical and basal poles. The drawback of that configuration is that apicobasal and basoapical vesicle traffic labelled at the opposite poles cannot be visualized independently because the same fluorescent marker has access to both poles. NEW METHOD: A double-barrel, capillary perfusion system was developed to independently stain either one pole or both the apical and the basal poles of isolated OHCs using different types of fluorescence membrane markers. RESULTS: Producing laminar fluid flow, the double-barrel perfusor allows investigation of the dynamics of apicobasal and basoapical vesicle traffic independently and/or simultaneously in the same OHC. COMPARISON WITH EXISTING METHOD: This method offers a unique option for investigating bidirectional vesicle traffic in bipolar epithelial cells, which is superior to other already established labelling techniques. CONCLUSIONS: The double-barrel perfusion system, suitable for selectively staining a longitudinal section of the plasma membrane of an isolated bipolar epithelial cell, opens new possibilities for investigating cell labelling and intracellular vesicle traffic.
