Fig 1.

pHi recovery after an acid-load (ammonium pre-pulse) in CCL39 wild-type (CCL39-wt) or mutant cells lacking NHE-1 (CCL39-nhe-1⁻). Exponentially growing non-neoplastic Chinese hamster lung fibroblast CCL39 cells seeded on glass cover slips were incubated for 30 min. in glucose/saline solution bicarbonate-free, HEPES-buffered solution adjusted to pHo 7.4. The pH-sensitive fluorescent dye 2′,7′ bis (carboxyethyl)-5-(6)-carboxyfluorescein (BCECF-AM) was then added to the cells for 5 min. before transfer of cells to a laminar flow cell chamber perfused with the same solution. Cells were maintained for 10 sec. and then a NH₄Cl solution (NH₄⁺) that causes alkalinization (ammonium pre-pulse) was added. The cells were shifted to a pHo 7.4 Na⁺/NH₄Cl-free solution (0 mM Na⁺) (containing choline chloride instead of NaCl), which causes intracellular acidification due to extrusion of NH₃ while preventing exchanger activity. Re-introduction of Na⁺ (120 mM Na⁺) allowed for exchanger activity. Ratiometric measurement of the fluorescence of 50 randomly selected individual cells per cover slip was performed in a workstation (Acquacosmos). The pHi was estimated by in situ two-point calibration (pHo 6.6 to 7.6) with perfusion of glucose/saline KCl/nigericin containing solutions to measure the pHi as a function of the fluorescence ratio.