Mitochondrial Dysfunction in BrainSpheres

Mitochondrial dysfunction was measured by MitoTracker Red CMXRos (Life Technologies, Carlsbad, CA, USA) following the protocol described in Pamies et al. (2018 (link)). Briefly, after 8 weeks of exposure to 0, 20 or 60 ng/ml of paroxetine, 10 BrainSpheres per condition were plated in 24-well-plates (500 μl). One microliter of MitoTracker Red CMXRos was added to the medium and incubated for 30 min at 37°C, 5% CO₂. The BrainSpheres were then washed twice and fixed with 4% paraformaldehyde (PFA) for 1 h and washed again twice with PBS. The Shandon Immuno-Mount (Thermo Fisher Scientific, Waltham, MA, USA) was used to mount the spheroids onto microscope cover slides (Thermo Fisher Scientific, Waltham, MA, USA). Images were taken using a Olympus BX60. The fluorescence was quantified using ImageJ software¹ and normalized to the size of the aggregates. To determine statistical significance, one-way ANOVA was performed with post hoc Bonferroni test. All data given are the means ± SD of three independent experiments performed with 10 technical replicates.