4.5. Measurement of Intracellular pH in DRG Cultures by Epifluorescence Microscopy with BCECF

DRG neurons were plated onto 24 mm round coverslips and were incubated with 1 μM BCECF (Life Technologies, Italy) in Krebs–Ringer Buffer (KRB, 135 mM NaCl, 5 mM KCl, 0.4 mM KH₂PO₄, 1 mM MgSO₄, 5.5 mM glucose, 20 mM 4-(2-hydroxyethyl)-1-piperazine ethane sulfonic acid (HEPES), pH 7.4) containing 2 mM CaCl₂. After 15 min (room temperature), the cells were washed and re-suspended in KRB (pH 7.4). A Leica DMI6000 epifluorescent microscope equipped with an S Fluor ×40/1.3 objective was used. Cells were alternatively excited at 490/450 nm (monochromator Polychrome IV, Till Photonics, Kaufbeuren, Germany), and the fluorescent signals were collected every 10 s (Hamamatsu, Shizuoka, Japan); the experiments were controlled and images analyzed using MetaFluor (Molecular Devices, Sunny-vale, CA, USA) software. To obtain the intracellular pH value, the 525/610 nm emission fluorescence ratios were compared with the calibration curves arising from the in vivo pH equilibration using the proton ionophore nigericin (10 µM) and Intracellular pH Calibration Buffer Kit (pH 7.5–5.5, Life Technologies, Italy).