Ratiometric Imaging of Intracellular pH

We measured intracellular pH in single cells by live-cell ratiometric imaging experiments using the fluorescent pH- sensitive BCECF dye^{48 (link)}. DRG cultures were incubated with 1 µM BCECF-AM (Life Technologies, Italy) for 20 min at room temperature in Tyrode standard solution (TS) of the following composition in mM: NaCl 154; KCl 4; CaCl₂ 2; MgCl₂ 1; 4-(2-hydroxyethyl)-1-piperazine ethane sulfonic acid (HEPES) 5; glucose 5.5; NaOH to pH 7.4. After washing in TS, cells were placed under a Leica DMI6000 epifluorescent microscope equipped with S Fluor × 40/1.3 objective and BCECF was alternatively excited at 490 nm and 450 nm (monochromator Polychrome IV, Till Photonics, Germany) while recording at emission wavelengths > 525 nm. Data was acquired every 3 s (Hamamatsu, Japan) with MetaFluor software (Molecular Devices, Sunny-vale, CA, USA). Image time series were analysed with ImageJ (Rasband W.S., NIH, Bethesda MD) and OriginPro 9.1 (OriginLab, USA) softwares to obtain the background subtracted ratio of the mean pixel intensity within a region of interest encompassing a neuronal cell body. The ratios were converted into the pH_i value by the high K⁺/nigericin technique. At the end of the experiment we obtained for each neuron three calibration points at pH values of 5.5, 6.5 and 7.5 (Calibration Buffer Kit, Life Technologies) that in almost all cells were best fitted by a straight line, whose parameters were used to convert ratios to pH values.