A simple method for monitoring changes in cell height using fluorescent microbeads and an Ussing-type chamber for the inverted microscope

Abstract

In this study, we report two developments for studies of ion transport in cultured epithelial cells. First, a convenient method is presented for measuring apparent cell height using fluorescent microbeads as high-contrast landmarks of the apical and basal cell surfaces. The apparent cell height is then used as an indicator to monitor the time course of changes in cell volume in response to osmotic perturbations. Second, an Ussing-type chamber design for the inverted fluorescence microscope is presented, which allows determination of transepithelial electrical properties. Using these two methods, we obtained simultaneous measurements of cell height and transepithelial electrical parameters for cultured renal (A6) epithelium. Cell height was measured by alternately focusing the microscope between microbeads marking the apical and basal surfaces. The distance between these two surfaces was measured electrically from the voltage output of a potentiometer that was mechanically coupled to the fine-focusing knob of the microscope. Following decreases in the bathing solution osmolality, the cell height and transepithelial Na+ transport rate (measured as short-circuit current, ISC) increased. The increase in cell height preceded changes in ISC by several minutes, suggesting a lack of direct linkage between changes in cell volume and transepithelial Na+ transport. Both the fluorescent microbead cell height method and the Ussing-type chamber can be used in conjunction with patch-clamp techniques, intracellular microelectrode impalements, or fluorescent probes of intracellular composition. Therefore, this system may be advantageous for studies of epithelial cell volume and channel regulation.