Measurement of intracellular pH in cultured cells by flow cytometry with BCECF-AM

This study evaluates the suitability of flow cytometry with the fluorochrome BCECF for measuring the intracellular pH (pH(i)) of cultured cells, and monitors the changes in pH(i) in murine hybridoma in batch culture and chick embryo fibroblast in monolayer culture (5th passage). The technique produced highly reproducible, repeatable results. The theoretical sensitivity from the calibration curve was 0.0004 pH units. But analysis of the standard deviation of the histogram of the green/red fluorescence ratios indicated a mean sensitivity of 0.08 (0.07-0.09) pH units. Interference due to cell size, fluorochrome incorporation and esterases were minimized by establishing a calibration curve with the cells whose pH(i) was to be measured using the 525/610 nm fluorescence ratio after excitation at 488 nm. The pH(i) of exponentially growing, batch cultured hybridomas was 7.50 at the start of culture. pH(i) increased during the exponential growth phase and dropped towards cell death. The pH(i) of the chick fibroblasts in monolayer culture was 7.30.