Quantifying Mitochondrial Morphology in Cortical Neurons

The NRF1mitoGFP reporter was a gift from K.J. Tronstad (University of Bergen, Bergen, Norway; Hodneland Nilsson et al., 2015 (link)). Cortical neurons were transfected with reporter-expressing plasmids on DIV 9. 24 h after transfection, cells were exposed to one of the following drug conditions: (1) vehicle (saline); (2) 1 µM atractyloside alone; or (3) atractyloside plus 5 mM NAC (Chao et al., 2016 (link)), followed by live-imaging for another 24 h in a 95% air/5% CO₂–gassed chamber mounted on a Leica TCS SP8 confocal laser scanning platform, equipped with Leica HyD hybrid detector and visualized through a HC PL APO CS2 63× (1.40 NA) oil-immersion objective. Image acquisition was controlled by LAS X. GFP was excited at 480 ± 15 nm and emission detected at 520 ± 15 nm. To analyze mitochondrial morphology the GFP signals of each cell (in a different set of cultures) were imaged at 24 h after drug treatment, and later, the Z-stack images were used to construct the 3D model by the LAS X for morphology analysis.