*3.7. Automated Microscopy and Image Analysis*

Plates were imaged with an Operetta high-content wide-field fluorescence imaging system, coupled to Harmony software (Perkin Elmer, Shelton, CT, USA). Wells in a 96-well plate were captured at 25 locations per well at 20× magnification at three wavelengths (350 nm: Hoechst 33,342; 554 nm: CellMask Plasma Membrane; 644 nm: MitoTracker Deep Red). The three images were combined and analyzed using the Harmony software. The analysis protocol involved in the following steps: (1) each cell nucleus was identified using Hoechst stain; (2) the cell cytoplasm as defined from CellMask fluorescence; (3) cells that overlapped the border of the image were excluded from the analysis. Each concentration was repeated in at least two separated experiments. To obviate observer bias, the image analysis was automated using the same parameters for every image.
