Bioscience Reports 3, 631-642 (1983) 631 Printed in Great Britain A novel method for measuring intracellular pH and potassium concentration C. L. BASHFORD, G. ALDER, K. 3. MICKLEM% and C. A. PASTERNAK Department of Biochemistry, St. George's Hospital Medical School, Cranmer Terrace, London SWI7 ORE, U.K. (Received 16 June 1983) The concentration of Na + and K + and the pH in the cytoplasm of Lettr~ cells was measured by monitoring the net flux of H +, Na +, or K + across the plasma membrane which had been rendered permeable to these ions by the action of Sendal virus. Ion flux was measured directly by analysis of cell composition, or indirectly by observing the change in membrane potential of cells treated with a specific ionophore. Cytoplasmic concentrations of cations were obtained by establishing the concentration of the cation in the medium at which addition of Sendal virus causes no change in cytoplasmic cation content. The value of Lettr~-cell pH was confirmed by direct measurement employing 3tp nuclear magnetic resonance, and the values of Na + and K + concentration were confirmed by analysis of cell cation and water content. Lettr~ cells suspended at 32~ in Hepes-buffered saline at pH 7.3 maintain a cytosolic pH of 7.0 and contain 30 mM Na + and 80 mM K +. Introduction Many recent reports have stressed the importance of monovalent cation content and ion fluxes across the plasma membrane in the regulation of cell growth and proliferation (Kaplan, 1978). Mitogenic transformation of lymphocytes, for example, is associated with modified cation 'metabolism' and is largely abolished by ouabain (Quastel & Kaplan, 1968), a specific inhibitor of the plasma- membrane (Na+/K+)ATPase ($kou, 1960, 1965). Indeed it is becoming clear that transformation and stimulation of many cell types is associated with changes in ion flux and in related parameters such as membrane potential and intracellular pH (Kiefer et al., 1980; Tsien et al., 1982; Gerson et al., 1982). A number of different methods are available for monitoring cytoplasmic ion contents (Morville et at., /973; Pietrzyk et al., 1978; Schummer & $chiefer, 1980) and intra- cellular pH (Gillies & Deamer, 1979; Hes, 1981). The procedures usually involve sophisticated methods for estimating the relative sizes of different, particularly extracellular, water spaces and do not usually address the problem of intracellular compartmentation directly. * Present address: Department of Biochemistry, University of Oxford, South Parks Road, Oxford OXl 3QU, U.K. 91983 The Biochemical Society