MEASUREMENT OF INTRACELLULAR PH IN CULTURED-CELLS BY FLOW-CYTOMETRY WITH BCECF-AM

This study evaluates the suitability of flow cytometry with the fluoro chrome BCECF for measuring the intracellular pH (pH(i)) of cultured ce lls, and monitors the changes in pH(i) in murine hybridoma in batch cu lture and chick embryo fibroblast in monolayer culture (5th passage). The technique produced highly reproducible, repeatable results. The th eoretical sensitivity from the calibration curve was 0.0004 pH units. But analysis of the standard deviation of the histogram of the green/r ed 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 fluoresc ence ratio after excitation at 488 nm. The pH(i) of exponentially grow ing, batch cultured hybridomas was 7.50 at the start of culture. pH(i) increased during the exponential growth phase and dropped towards cel l death. The pH(i) of the chick fibroblasts in monolayer culture was 7 .30.