To assay the electrochemical potential across the IMM of cultured cells, we used the cell-permeant dye TMRM. For colocalization studies with Mt-GFP, TMRM was used at 20 nM on DIV14 and incubated for 30 min before imaging. For the orthogonal screen of compounds, TMRM was added to primary neurons on DIV13 at 10 nM, 1 hour before pintooling selected compounds, and incubated for 24 hours. Cells were washed 2× with prewarmed fresh feeding media without B27 before measurements with a plate reader.

The cell-permeable dye MitoTracker Orange (ThermoFisher) was used to label mitochondria in live primary neurons according to the manufacturer’s protocol. Briefly, neurons at DIV14 were stained with 50 nM MitoTracker Orange dissolved in fresh feeding media and incubated for 30 min. The cells were washed 2× with fresh feeding media before imaging.

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