In vivo mitochondrial pH measurement

Mitochondrial pH in live-cell was detected by using ratiometric pH sensor, SypHer-mito Imaging of mitochondrial pH was performed based on the method described in the original report with some modifications(Poburko et al., 2011). HeLa cells transfected SypHer-mito were grown on No. 1.5 glass coverslips and mounted in a stage-top incubation system (Tokai Hit) warmed to 37°C. Hanks’ balanced salt solution (HBSS, 21-023-CV, Corning) was used during image acquisition using live-cell pH measurement. All images were collected with a Nikon Ti inverted microscope equipped with 60× Plan Apo NA 1.4 objective lens and the Perfect Focus System for continuous maintenance of focus. For pH imaging, pairs of fluorescence images were obtained using SypHer-mito. For detecting pH-sensitive fluorescence change, SypHer-mito was excited at 480nm (selected with a HQ480/40× filter, Chroma) and collected with a long-pass dichroic mirror (Q505lp, Chroma), and a 535/50 emission filter (ET535/50m, Chroma). For detecting internal standard fluorescence, SypHer-mito was excited at 430nm (selected with an ET430/24× filter, Chroma) and collected with a dual band pass dichroic mirror (69008bs, Chroma), and a 535/30 emission filter (ET535/30m, Chroma). Images were acquired with a Hamamatsu ORCA-R2 cooled CCD camera controlled with MetaMorph 7 software. For timelapse experiments, pairs of images were collected every 1 min, using an exposure time of 500 ms with illumination light shuttered between acquisitions. Cells were preincubated in HBSS for 30 min. After that, images for basal pH measurements were acquired for 10 min, then cells were treated with 0.5 μM CCCP and traced the pH change for 15 min. Fluorescence ratios (F480/F430) were calculated in ImageJ 1.49 (NIH) and analyzed in Excel (Microsoft) and GraphPad Prism 6 (GraphPad). After each experiment, mitochondrial pH was calibrated using nigericin (5 μg/mL) and monensin (5 μM) in 125 mM KCl, 20 mM NaCl, 0.5 mM MgCl2, 0.2 mM EGTA, and 50 mM HEPES (pH 8.0), PIPES (pH 6.5 – 7.5), and MES (pH 6.0). Absolute pH value was calculated by fitting to the calibration curve.