Average intracellular pH( pH i) was measured in Chinese hamster ovary (CHO) and murine NG 108-15 neuroblastoma cells by the weak acid,$[{}^{14}{\rm C}]5,5\text{-dimethyl-}2,4\text{-oxazolidinedione-}2\ ([{}^{14}{\rm C}]{\rm DMO})$. Intercell variance in pH i in CHO cells was determined by flow cytometry (FCM) and automated image analysis using the pH-sensitive fluorescent dye, 2′,7′-bis-(carboxyethyl)-5(and-6)-carboxyfluorescein. Our results are summarized as follows: (i) When extracellular${\rm pH}({\rm pH}_{{\rm e}})$ ranged from 6.1 to 7.9, the relationship between pH e and average pH i was similar for both cell lines, except that pH i of NG 108 cells was 0.3 to 0.2 pH units higher than that of CHO cells. (ii) When both cell lines were heated at 43.5°C to give an isosurvival of 0.1 at pH e 7.2, lowering${\rm pH}_{{\rm e}}\ (6.1-6.9)$ reduced survival more for CHO cells than for NG 108-15 cells. However, plots of survival versus average pH i were identical for the two cell lines. (iii) Values of average pH i measured by$[{}^{14}{\rm C}]{\rm DMO}$ agree with those measured by FCM techniques. (iv) A distribution of pH i values was obtained for a population of cells. However, when the cells were sorted on the basis of low or high values of pH i and reanalyzed, the distributions of the sorted populations were almost identical to the distribution of the original population. These results indicated that the distribution of pH i for a cell population is much more homogeneous than that observed by FCM. (v) These observations indicate that pH i at the time of heating and not pH e is responsible for the low pH sensitization of heat killing.

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