Cardiac Voltage-Sensitive Dye Imaging

Cells were washed 5 times with indicator-free Tyrode’s solution. After reconstituting the 2 mM VF2.1Cl dye to 1 mM in 10% Pluronic F-127, then diluting the dye to 100 nM in Tyrode’s solution, the VF2.1Cl dye (MilliporeSigma) was added to each well for 50 minutes in a 37°C 5% CO₂ incubator. The cells were then washed 5 times with indicator-free Tyrode’s solution and returned to the 37°C 5% CO₂ incubator for 10 minutes to recover. The dye was excited at 514 nm wavelength, and time series images were acquired at an acquisition frequency of 45 Hz for 40 seconds in Epi-fluorescence mode using Zeiss Laser TIRF Microscope fitted with a Hamamatsu ORCA-Flash 4.0 V3 digital CMOS camera C13440-20CU. HMR-1556 (Tocris) was added at a concentration of 1 μM 30 minutes prior to image acquisition and incubated in a 37°C 5% CO₂ incubator. APD₉₀ was calculated as described previously (55 (link)).