Quantitative High-Content Imaging Cytotoxicity

Cytotoxicity and mitochondrial integrity were quantitatively assessed by high-content imaging after conclusion of Ca²⁺ flux measurements at 90 min and prior to TempO-seq lysate preparation at 24 hrs as described previously (Grimm et al., 2016 (link)). Briefly, cell culture medium including (90 min) or excluding (24 hrs) Ca²⁺ dye was replaced with 25 μl of staining solution (2 μg/ml Hoechst 33342 and 0.2 μM MitoTracker Orange in iCell cardiomyocyte maintenance medium). Following 15 min incubation at 37°C and 5% CO₂ the staining solution was discarded and replaced with an equal volume of fresh maintenance medium. Images were then acquired using an ImageXpress Micro Confocal cellular imaging system (Molecular Devices), using DAPI and Cy3 filters for Hoechst 33342 and MitoTracker Orange, respectively. Images were processed using the instrument-specific MetaXpress software package (Molecular Devices). Quantification of imaging-based parameters for concentration-response assessment was achieved using the multi-wavelength cell scoring application module.