Fluo-4 AM Calcium Imaging of Cardiomyocytes

Fluo-4-acetoxymethyl ester (fluo-4 AM) (Invitrogen, ThermoFisher Scientific) was used to visualise the intracellular calcium transients. NRVMs were loaded with 4 μL fluo-4 AM (prepared as 50 µg Fluo-4 AM dissolved in 50 µL DMSO) in 1 mL DMEM and placed in the incubator at 37 °C for 20 min^{11 (link),20 (link)}. The DMEM was refreshed, and the cells were returned to the incubator for a further 20 minutes for de-esterification of the dye. The constructs were mounted on the stage of an upright Nikon Eclipse FN1 microscope or an inverted Nikon Eclipse TE2000 microscope in a glass bottom dish (MatTek Corporation) and observed through a 40x water immersion or 40x oil objective respectively. Ca² transients in cardiomyocytes were studied by field stimulating the cells at 1 Hz to induce rhythmic depolarisation, and line scans were recording. A custom made MATLAB code was used to calculate the normalized amplitude as f/f0, time to peak (Tp), times to 50% (T50) and 90% declines (T90) in the transients^{28 (link)}.