Imagexpress micro confocal cellular imaging system

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.

After conclusion of the Ca²⁺ flux measurements at 90 minutes post-chemical addition, cell viability-related phenotypes were quantitatively assessed through high-content imaging with the ImageXpress Micro Confocal Cellular Imaging System (Molecular Devices) as previously reported ^{11 (link), 36 (link)}. The high-content imaging assay was performed as follows. First, the total volume of cell culture medium containing Ca²⁺ dye was aspirated and replaced with 25 µL/well pre-warmed staining solution with fluorescent probes for nuclei and mitochondria (2.2 µg/mL Hoechst 33342 and 0.2 µM MitoTracker Orange in cardiomyocyte maintenance medium with 1:500 penicillin/streptomycin solution). Microplates were then incubated at standard conditions for 15 minutes, the staining solution was aspirated, and 25 µL/well pre-warmed fresh maintenance medium containing 1:500 penicillin/streptomycin solution was added to each well to wash before proceeding to image acquisition. Images of each well at 10× magnification were acquired in succession using DAPI (Hoechst 33342 for nuclear staining), TRITC (MitoTracker Orange for mitochondrial staining), and FITC (Ca²⁺ dye) filters.