C1272

Cells were seeded at 70,000 per well in 4-chamber Nunc Lab-Tek slides (734-2060, ThermoScientific). Experiments involving superfusion were performed on a Leica LCS confocal microscope to image fluorescence. Most experiments measured intracellular pH using cSNARF1 (17 μM for 10 min; C1272 ThermoFisher), excited at 514 nm and collected at 580/640 nm. The ratio was calibrated to units of pH using a calibration curve determined in separate experiments using the nigericin technique [21 (link)]. Solutions were delivered by a system of tubes operated by a peristaltic pump, with a two-level solution switcher to alternate between one of two superfusates heated to 37 °C. Excess solution was drawn by a vacuum pump to ensure laminar flow. Solutions were based on normal Tyrode containing 4.5 mM KCl, 1 mM CaCl₂, 1 mM MgCl₂, 11 mM glucose, 10 mM HEPES, 10 mM MES and 130 mM NaCl, titrated to the desired pH (5.7–7.7) using HCl or NaOH.

The cytosolic pH was determined with SNARF®-1 (5-(and-6)-Carboxy SNARF™-1, acetoxymethyl ester, acetate; C1272, ThermoFisher), which is suitable for ratiometric pH measurements. SNARF®-1 was excited at 488 nm and emissions were recorded at 580 nm and 640 nm, essentially as described 73 . For calibration, U251N cells were seeded in 96-well plates, containing 10,000 cells/well. After overnight growth, samples were incubated with 5 μM SNARF®-1 in serum-free/phenol-free DMEM (45 min, 37^oC). Calibration was performed for different pH values (pH 5.5, 6.0, 6.5, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5) in the presence of 10 μM nigericin (Sigma) 73 . Fluorescence intensities were measured with a SPARK10M microplate reader (excitation: 485 nm, bandwidth 20 nm; emission E₁: 635 nm, bandwidth 35 nm; and emission E₂: 580 nm, bandwidth 20 nm). The ratio E₁/E₂ for fluorescence emission was plotted as a function of pH, and non-linear regression was used for curve-fitting. The cytosolic pH values of U251N cells were extrapolated from the calibration curve.