Comparison of DMO and flow cytometric methods for measuring intracellular pH and the effect of hyperthermia on the transmembrane pH gradient

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Abstract
Intracellular pH (pHi) was measured in both unheated and heated cells by the distribution of the weak acid, 5,5‐dimethyl‐2,4‐oxazolidinedione‐2‐14C (14C‐DMO), and by the fluorescence intensity ratio (I530/I630) of the pH sensitive fluorescent dye, 2′,7′‐bis(carboxyethyl)‐5,6‐carboxyfluorescein (BCECF), analyzed by flow cytometry (FCM). BCECF‐loaded Chinese hamster ovary (CHO) cells were analyzed by FCM after they had incubated in fresh medium at 37°C for 90 min, during which time a decrease in fluorescence ratio stabilized. After stabilization, the pHi determined for CHO cells by the FCM method at pHe values of 6.0–8.1 agreed‐within 0.1 pH units with that determined by the 14C‐DMO method. There is a pH gradient across the plasma membrane that is not affected by heat. In CHO cells, the gradient, determined by DMO and FCM, is less or greater than pHe by 0.30 and 0.15 pH units at pHe 7.4 and 6.3, respectively, and in NG108‐15 cells, the gradient determined by DMO increases to 0.50 pH units at pHe 6.3. Both cells maintained their pH gradients for at least 4 h after heating, although 99.9% of the cells were reproductively dead (survival of 10−3) after heating at 45.5°C either at the normal pHe of 7.4 or at a low pHe of 6.4–6.7.