Simultaneous detection of cell volume and intracellular pH in isolated rat duodenal cells by confocal microscopy and BCECF

Abstract

The combination of confocal laser scan microscopy and the pH-sensitive fluorescent dye BCECF allowed us to record simultaneously intracellular pH, cell viability and relative cell volume. pH was measured by using the pH-sensitive excitation wavelength at 488 nm and the pH-independent excitation wavelength at 442 nm to obtain ratio images. Cell volume was traced by measuring fluorescence dye concentration at 442 nm. Isolated villus tip rat duodenal enterocytes were exposed to 20 mM NH₄Cl, sodium free, or 1 mM amiloride buffer. Sodium free buffer and amiloride buffer acidified the cells. Cell volume did not change in sodium free buffer, or NH₄Cl exposure, but amiloride led to an increase in cell volume of 20%. After acidification of the duodenal cells, amiloride buffer increased cell volume by almost 50%. These studies revealed that cell volume regulation during pH changes in short-living cells could easily be detected by confocal microscopy and BCECF.