2.9. Nonyl Acridine Orange (NAO) Staining

NAO staining has been used to measure mitochondrial content in cells. The dye binds specifically to cardiolipin in mitochondria. Neurons were seeded in a 6-well-plate and transfected as detailed above. NAO dye (400 nM, Sigma Aldrich, St. Louis, MO, USA) was added to cells at 14–16 DIVs in mKRB. Cells were stained for 30 min at 37 °C, washed with mKRB, detached mechanically by a soft scraping and suspended in mKRB at the concentration of 100,000 cells/mL. Mitochondrial mass was assessed by flow cytometry using the FACS Canto II flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA).