Calcium Transient Measurement Protocol

Calcium transients were assessed using an EarlyTox Cardiotoxicity Kit (Molecular Devices, San Jose, CA, USA) (28 (link)). Calcium dye loading was performed according to the manufacturer’s instructions. EarlyTox calcium dye was resuspended in the supplied buffer and added to the cells in a 1:1 ratio with the CM maintenance media. The plate was incubated for 2 h (37°C, 5% CO₂) before recording calcium transients for 2 min at 37°C on the FLIPR Tetra System (Molecular Devices), using the following parameters: excitation, 470–495 nM; emission, 515–575 nM; exposure time, 50 ms; light-emitting diode intensity, 50%; and interval time, 100 ms. Calcium traces were produced and analyzed using SoftMax Pro Software (Molecular Devices).