Cellcarrier 96 well special optics plates

HPSC-cardiomyocytes were seeded as full monolayers (30 K cells per well) onto vitronectin-coated CellCarrier-96 well special optics plates (PerkinElmer) and were treated with 1 μM of the anticancer drugs DOXO, AMR or ACLA 10-12 days after seeding. Images of hPSC-cardiomyocytes were acquired using the high-throughput automated EVOS FL Auto 2 (Thermo Fisher) microscope equipped with a 40x Super-apochromat Olympus objective (NA 0.95) (Thermo Fisher, AMEP4754). Anthracyclines were quantified based on auto-fluorescence using the RFP filter cube (ex531/20nm; em593/40nm, Thermo Fisher, AMEP4652). The cell culture area of the well was scanned by automatically acquiring 55 images per well at indicated timepoints. Cells were maintained during the 24 hours at 37°C and 5% CO₂ on the EVOS FL Auto 2 with the EVOS Onstage incubator (Thermo Fisher). For flow cytometry quantification, cardiomyocytes were treated in suspension with the compounds and autofluorescence was quantified (at ex561nm, em586/15) with a MACSQuant VYB flow cytometer (Miltenyi Biotech) at the indicated time points. Plots were analyzed with FlowLogic software.

HiPSC-cardiomyocytes were seeded as monolayers onto vitronectin-coated CellCarrier-96 well special optics plates (PerkinElmer) in TDI medium and treated with the anthracyclines 10-12 days after seeding at the indicated concentrations. To quantify calcium kinetics and accumulation, cardiomyocytes were stained with the Fluo4-AM calcium dye (Thermo Fisher Scientific) at a final concentration of 1 μM with Pluronic (0.2 mg/ml) for 30 min at 37°C. Imaging was performed 24 hours or 2 weeks after treatment at 37°C and 5% CO₂ on a Nikon Eclipse TE2000-U Microscope with a 20x objective. Videos were taken at 70 fps, which were used to quantify Ca²⁺ fluxes into and out of the cell over time using a custom software script. The individual frame at the lowest fluorescent intensity (= complete relaxation of the cells) was used to determine the baseline Ca²⁺ load of the cardiomyocytes after drug treatment by measuring mean fluorescent intensity of the entire frame using ImageJ software.