BCECF in single cultured cells: Inhomogeneous distribution but homogeneous response

Using confocal laser scanning microscopy with a dual-wavelength laser system, the behaviour of BCECF [(29,79-bis-2-carboxyethyl)-5-(and-6)carboxyfluorescein] was investigated in a variety of cell lines. Selection of a small area for monitoring allowed discrimination between various intracellular organelles, whose identity was established by vital staining. It was found that, after loading the cells with BCECF, both the nucleus and the mitochondria showed a higher level of fluorescence than the cytoplasm. Calibration of the pH-sensitivity of these fluorescence signals using the nigericin method yielded identical curves, as did exposure of the cells to NH4Cl. These studies suggest that BCECF, despite its inhomogeneous intracellular distribution, reports the pH of only one cellular compartment, the cytosol.