HiPSC‐CMs were incubated in serum‐free medium (BMCC: CaCl2 1.49 mM, MgSO4*7H2O 0.81 mM, KCl 4.4 mM, NaHCO3 36 mM, NaCl 77.59 mM, Na2HO4P 0.91 mM, Na2SeO3‐5H2O 0.0001 mM, KNO3 0.0008 mM, d ‐glucose 25 mM, sodium pyruvate 1 mM) for at least 1 h and subsequently loaded with FluoVolt Dye (1:1000, Invitrogen, Cat# F10488) and Powerload Concentrate (1:100, Invitrogen, Cat# F10488) for 20 min at 37°C. Action potentials (APs) were recorded on the CellOPTIQ system (Hortigon‐Vinagre et al. 2016 (link)) using a 40× objective, 470 nm LED, photomultiplier tube (PMT) and a sensor with an acquisition rate of 10 kHz. Plates were placed in an on‐stage incubator during the experiment, maintaining 5% CO2 and 37°C. APs were analysed using CellOPTIQ software, which computed the time‐averaged APs (n > 3) and calculated the beating frequency, depolarization time (TRise) and AP duration at 90% of the amplitude (APD90) (Fig. 1A ).
Fluovolt dye
Manufactured by Thermo Fisher Scientific
Sourced in United States
The FluoVolt Dye is a fluorescent probe designed for labeling and detecting proteins in live cells. It functions by binding to cellular proteins, allowing for the visualization and quantification of protein expression and localization using fluorescence microscopy or flow cytometry techniques.
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6 protocols using fluovolt dye
1
Electrophysiological Characterization of hiPSC-Derived Cardiomyocytes
2
Fluorescent Voltage Imaging of hiPSC-CMs
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Cultures of hiPSC-CMs (5–6 days post-plating) were treated with FluoVolt dye (1:1000, Invitrogen, Cat# F10488) in SF media and incubated for 20 min at 37 °C with 5% CO2 and 80% humidity. Multi-well plates were placed in the environmentally controlled stage incubator (37 °C, 5% CO2, water-saturated air atmosphere) (Okolab Inc. Burlingame, CA) of the CellOPTIQ® platform (Clyde Biosciences Ltd., Glasgow, Scotland). The FluoVolt signal was recorded from a 0.2 mm × 0.2 mm area using a 40× (numerical aperture: NA 0.6) Excitation wavelength was 470 ± 10 nm using a light-emitting diode (LED), and emitted light was collected by two photomultipliers (PMTs) at 510–560 nm (channel 1) and 590–650 nm (channel 2), respectively. LED, PMT, and associated power supplies and amplifiers were supplied by Cairn Research Ltd. (Kent, UK) and digitized at 10 kHz and stored on a computer hard drive. In the case of FluoVolt-based action potential recordings, the short wavelength (channel 1, 51–560 nm) was analyzed for action potential features. A summary of the CellOPTIQ® configuration is shown in Figure 1 .
3
Live Imaging of Heart Organoids
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Prepare labeling solution by adding FluoVolt™ dye (Invitrogen) into RPMI + B27 medium (1:50), and remove old medium from human heart organoid cultures. After washing heart organoids with PBS twice, organoids were treated with 100 μl of labeling solution under room temperature for 20 min. After removing dye solution and washing with PBS twice, organoids were kept in a 96-well U-bottom culture plate with 100 μl of PBS. The resulting cells were then live imaged in a Leica DMi-8 microscope.
4
Strobe Photography for Membrane Dynamics
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The method of strobe photography, aimed at resolving membrane charging and relaxation kinetics with nanosecond resolution, was introduced and validated recently [34 (link)]. In the present study, strobe photography was performed using the same equipment and protocols as described in our previous report [17 (link)]. In short, CHO cells were loaded with a FluoVolt dye (Thermo Fisher Scientific) and illuminated by ~6 ns, 440 nm laser flashes, which were delivered in synchrony with nsEPs. The time interval between a laser flash and nsEP was decreased or increased in 50 ns steps, and one photo of the stimulated cell was collected at each time interval. The 11 µs long plots in Figure 5 were built from 220 individual photos. The nsEP amplitude was set at a level below the electroporation threshold to avoid membrane damage and ensure a similar response to multiple stimuli. Dye bleaching and the variability of laser power were compensated by averaging the data collected with time interval increments and decrements and by normalizing the emission changes at the cell poles to the whole-cell emission [17 (link)].
5
Cardiomyocyte Action Potential and Calcium Dynamics
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To record the action potentials of cardiomyocytes, cells were loaded with 1× FluoVolt dye as per manufacturer's instructions (F10488, ThermoFisher) and exchanged into phenol red free RPMI (11835030, ThermoFisher), supplemented to 1 mM calcium chloride. For calcium transient recordings, Cal 520 AM dye (21130, AAT Bioquest) was incubated at 2.5 μM for 30 min at 37 °C. Cardiomyocytes were placed in the Nikon Eclipse Ti2-E Inverted Microscope, fitted with a Nikon Plan Fluor 10× objective (NA, 0.3) and imaged using an Andor Zyla sCMOS (Oxford Instruments) high-speed camera. Data were collected at 5 ms temporal resolution from regions of 512 × 512 pixels. Before recording, cells were placed into the live chamber (37 °C and 5% CO2) and left to equilibrate for 30 min. Data were processed utilizing an in-house MATLAB script [33 (link)].
6
Optical Electrophysiology of Cardiomyocytes
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Optical-electrophysiology was performed as outlined previously73 (link) using a voltage sensitive FluoVolt dye (Thermo Fisher Scientific, USA), and the absolute fluorescence signals were obtained with a custom built novel high-throughput imaging platform (OptioQUANT) (Ternion Bioscience, Singapore). All data were analyzed with Igor Pro (WaveMetrics, USA). AP durations of WT and Mfn 1&2 KO mouse cardiomyocytes were measured at 30%, 50%, 70% and 90% decrement of AP amplitude and analyzed. Cells which exhibited irregular events during acquisition were excluded from analyses. All values are given as mean ± SEM.
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