Intracellular H+ imaging

To measure the intracellular H⁺ concentration ([H⁺]_i) changes in cultured cortical astrocytes, we used a confocal imaging system and acetoxymethyl ester of a proton‐sensitive dye, BCECF‐AM. The dye was loaded into the cells by incubating them with 3 μm BCECF‐AM in Hepes‐buffered solution for 15 min at room temperature. Cells were then mounted on a chamber under a confocal laser scanning microscope (LSM 510, Zeiss, Oberkochen, Germany) and superfused continuously either with Hepes‐buffered solution (in mm): NaCl 140, KCl 3, α‐d‐glucose 2, NaH₂PO₄ 0.5, Hepes 10, MgCl₂ 1, CaCl₂ 2; or CO₂/HCO₃ ⁻‐buffered solution (in mm): NaCl 114, KCl 3, NaH₂PO₄ 0.5, α‐d‐glucose 2, NaHCO₃ 26, MgCl₂ 1, CaCl₂ 2. BCECF was excited consecutively at 488 nm (proton‐sensitive wavelength) and 458 nm (close to the isosbestic point), and the changes in fluorescence emission were monitored at > 505 nm (using an LP 505 filter). Images were obtained every 5 s (0.2 Hz) with a 40× water immersion objective. The fluorescence emission intensity of 488 nm excitation changes inversely with a change in [H⁺]_i, whereas the fluorescence emission intensity of 458 nm excitation is largely pH‐insensitive. The changes in [H⁺]_i were monitored using the ratio F ₄₅₈/F ₄₈₈. This ratio was converted into pH and absolute intracellular proton concentrations ([H⁺]_i) using the nigericin‐based calibration technique. Cells were perfused with calibration solutions, titrated to pH 6.0, 6.5, 7.0 or pH 7.5, containing nigericin 10 μm, NaCl 15 mm, KCl 130 mm, Hepes 20 mm, MgCl₂ 1 mm and CaCl₂ 1 mm. Mean ratio values (F ₄₅₈/F ₄₈₈) were plotted as a function of pH to create the calibration curve. The ratio was then converted to pH_i and H⁺]_i (Theparambil et al. 2014, 2015). H⁺ buffering strength was calculated from the amplitude of the pH shift induced by addition of 5% CO₂ in the absence of external sodium to suppress the majority of acid/base transporter activity, as described in detail by Theparambil & Deitmer (2015).

Acute cortical slices were loaded with 4 μm of BCECF‐AM in aCSF saline for 30 min at 30°C. The slices were mounted in a perfusion chamber of an upright microscope (BX50WI; Olympus, Tokyo, Japan) equipped with an epifluorescence unit (Polychrome IV; Till Photonics, Gräfelfing, Germany). The slices were superfused continuously with aCSF buffered with 5% CO₂ containing (in mm): NaCl 116, KCl 2.5, α‐d‐glucose 10, l‐lactate 1, NaHCO₃ 26, NaH₂PO₄ 1.25, MgCl₂ 1 and CaCl₂ 2. The temperature of the aCSF was maintained at 34°C during experiments. BCECF was excited at 440 and 490 nm for 5 ms at an interval of 5 s. The 535 nm fluorescence emission of the two excitation wavelengths was monitored through a 40× water immersion objective with a Peltier‐cooled CCD camera (Till Photonics). The ratio of BCECF emissions, F ₄₄₀/F ₄₉₀, was converted into proton concentration using nigericin‐based calibration method as described before (Theparambil et al. 2014).