Cells were transfected with gCaMP6F [Addgene (81)] and imaged the next day. The mitochondrial membrane potential (Δψm) was assessed with 10 nM mitochondrial voltage-sensitive dye TMRE (Life Technologies) (82). After loading for 15 min at 37°C, the medium was exchanged to imaging buffer without TMRE, and cells were left to equilibrate at room temperature for 15 min. The imaging buffer consisted of 1.25 mM CaCl2, 19.7 mM Hepes, 4.7 mM KCl, 1.2 mM KH2PO4, 1 mM MgSO4, 130 mM NaCl, and 5 mM dextrose (pH 7.2) at room temperature. Cells were imaged using the excitation wavelengths of 488 nm (gCaMP6F) and 561 nm (TMRE), and average fluorescence intensities over 30 min were acquired. As a positive control for loss of Δψm, cells were treated with 50 μM FCCP (Life Technologies) to depolarize mitochondria at the end of the experiment. Cells were imaged at room temperature on a Leica SP5 microscope with a 63× 1.4 numerical aperture oil objective and 2.5× zoom. Fluorescence intensities were quantified with a combination of Leica TCS5 software and ImageJ [National Institutes of Health (NIH)].

For measurement of superoxides generated by mitochondria, cells were incubated with MitoSOX (Life Technologies) according to the manufacturer’s recommendations. Cells were then washed and imaged.

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