Flow cytometric measurement of intracellular pH in B16 tumors: intercell variance and effects of pretreatment with glucose.

Flow cytometry was used to measure cytoplasmic pH (pHi) of B16 melanoma cells taken from tumor-bearing animals. We used a ratiometric method to allow measurements on an individual cell basis which were independent of cellular content of the pH indicator BCECF. In order to "freeze" any intercell variance which may have existed within the tumor mass, tumors were mechanically disaggregated in bicarbonate-free medium containing 0.5 mM amiloride at 4 degrees C and loaded with BCECF in choline chloride-based Earle's solution at 37 degrees C. Studies using cells grown in vitro showed that this protocol prevented acid load recovery during the 30-min period typically required between tumor excision and pHi measurement. A calibration curve was obtained by resuspending BCECF-stained cells in a range of buffers containing the proton ionophore nigericin. The range of values for individual cells was estimated by comparing the coefficient of variation of the test sample with that obtained when nigericin was used to reduce all cells to the pHi of the calibration buffer. The average value for mean tumor cell pH was 7.32 +/- 0.05 SD. Pretreatment of animals with intraperitoneal glucose for one hour resulted in an average for mean pHi of 7.17 +/- 0.17 SD. Mean coefficient of variation was 8.7%, and in the presence of nigericin, 8.1%. These values indicate a variance in measured pHi of approximately +/- 0.4 pH units, but most of this results from experimental error rather than true intercell pHi variance. The method used here is capable of detecting reduction in mean tumour pHi caused by ip glucose, but incapable of precise estimation of individual cell values. Despite these uncertainties, the results suggest that the range of pHi within B 16 tumors is small.