Name: single-cell optical action potential measurement in human induced pluripotent stem cell-derived cardiomyocytes
Uploaded: 2020-12-22T13:30:00
Description: Watch this Scientific Journal Video about Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes at JoVE.com

The authors would like to acknowledge Cairn Research Ltd. for their kind financial contribution which covered production costs of this publication. In addition, we thank Ms. Ines Mueller and Ms. Stefanie Kestel for their excellent technical support.
The authors&#8217; research is supported by the German Center for Cardiovascular Research (DZHK), the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation, VO 1568/3-1, IRTG1816 RP12, SFB1002 TPA13 and under Germany&#8217;s Excellence Strategy - EXC 2067/1- 390729940) and the Else-Kr&#246;ner-Fresenius Stiftung (EKFS 2016_A20).

1. Cellular preparations
NOTE: Human iPSCs used in this protocol were derived from healthy donors and differentiated in monolayers using fully defined small molecule modulation of WNT signaling and lactate purification techniques as previously described12,13,14. iPSC-CMs were maintained every 2-3 days with a culture medium outlined below.
<ol>
	<li>Prepare a culture medium of basal medium (RPMI 1640...

<ol>
	<li>Miller, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+molecule+fluorescent+voltage+indicators+for+studying+membrane+potential.">Small molecule fluorescent voltage indicators for studying membrane potential.</a> Current Opinion in Chemical Biology. 33, 74-80 (2016).</li><li>Liang, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drug+screening+using+a+library+of+human+induced+pluripotent+stem+cell-derived+cardiomyocytes+reveals+disease-specific+patterns+of+cardiotoxicity.">Drug screening using a library of human induced pluripotent stem cell-derived cardiomyocytes reveals disease-specific patterns of cardiotoxicity.</a> Circulation. 127 (16), 1677-1691 (2013).</li><li>Horv&#225;th, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low+resting+membrane+potential+and+low+inward+rectifier+potassium+currents+are+not+inherent+features+of+hiPSC-derived+cardiomyocytes.">Low resting membrane potential and low inward rectifier potassium currents are not inherent features of hiPSC-derived cardiomyocytes.</a> Stem Cell Reports. 10 (3), 822-833 (2018).</li><li>Salama, G., Morad, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Merocyanine+540+as+an+optical+probe+of+transmembrane+electrical+activity+in+the+heart.">Merocyanine 540 as an optical probe of transmembrane electrical activity in the heart.</a> Science. 191 (4226), 485-487 (1976).</li><li>Hortigon-Vinagre, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+ratiometric+fluorescence+measurements+of+the+voltage+sensitive+dye+Di-4-ANEPPS+to+examine+action+potential+characteristics+and+drug+effects+on+human+induced+pluripotent+stem+cell-derived+cardiomyocytes.">The use of ratiometric fluorescence measurements of the voltage sensitive dye Di-4-ANEPPS to examine action potential characteristics and drug effects on human induced pluripotent stem cell-derived cardiomyocytes.</a> Toxicological Sciences. 154 (2), 320-331 (2016).</li><li>Blinova, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=International+multisite+study+of+human-induced+pluripotent+stem+cell-derived+cardiomyocytes+for+drug+proarrhythmic+potential+assessment.">International multisite study of human-induced pluripotent stem cell-derived cardiomyocytes for drug proarrhythmic potential assessment.</a> Cell Reports. 24 (13), 3582-3592 (2018).</li><li>Miller, E. 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Here we describe a basic protocol to easily acquire detailed AP profiles from isolated iPSC-CMs suitable for electrophysiological modelling and cardiac drug screening. We detect regular, robust APs from our sparsely seeded iPSC-CMs which suggests both indicator functionality and methodological fidelity.
Due to the wide spectrum of commercial methodologies for iPSC reprogramming and lack of standardization for cardiac differentiation protocols, iPSC based models can show immense variability in ...

Electrophysiological modeling of cardiomyocytes and the construction of efficient platforms for cardiac drug screening is essential for the development of therapeutic strategies for a variety of arrhythmic disorders. Rapid expansion of induced pluripotent stem cell (iPSC) technology has produced promising inroads into human disease modelling and pharmacological investigation using isolated patient derived cardiomyocytes (iPSC-CM). &#8220;Gold standard&#8221; techniques for electrophysiological characterization of these cells through patch-clamp (current-clamp) can quantify action potential (AP) morphology and duration, however, this method is incredibly complex and slow, and not well suited for high throughput data acquisition1. iPSC-CMs are regularly reported to have an increased diastolic membrane potential and increased leak current when compared to adult native cardiomyocytes2. It is suggested that smaller cell size and reduced membrane capacitance observed in iPSC-CMs may produce some systematic error when using the current-clamp technique, perhaps explaining these deviations3. In order to maximize the usefulness of an iPSC-CM platform, an additional method is valuable to increase throughput and ensure data accuracy when characterizing transmembrane voltage changes at a single cell level in iPSC-CMs.
Voltage sensitive dyes (VSD) have long been a proposed method to provide faster, non-invasive and equivalent analysis of cardiac AP kinetics comparative to those of traditional techniques4. A recent study has demonstrated the suitability of ratiometric voltage sensitive probe photometry to accurately quantify the cardiac AP5. Furthermore, the ability to readily scale up optical photometry approaches lends this technique to large scale cardiotoxicity screens critical in therapeutic drug development (e.g., CiPA). Development of standardized cardiotoxicity protocols in a blinded multi-site study using microelectrode array and voltage-sensing optical techniques has demonstrated the key value of this approach6.
Many potentiometric dyes are commercially available, and ongoing synthetic development of new probes show exciting potential for streamlining their effectiveness across a variety of cardiac and neural constructs. The ideal VSD will have augmented kinetics and sensitivity, while displaying decreased capacitive load, photobleaching and cytotoxicity. The recently synthesized VF2.1Cl (FluoVolt) expresses many of these beneficial properties largely due to its novel wire-based molecular structure, shared by other members of the new VoltageFluor (VF) family7. In contrast to common electrochromic VSDs in which simple probes molecularly and electrically conjugate to the plasma membrane, this dye consists of a passively inserted, membrane-spanning synthetic wire which pairs an electron-rich donor with a modified fluorescein fluorophore (FITC). Mechanistic details are provided in Figure 1. This dye demonstrates excellent sensitivity to membrane voltage fluctuations, displaying a 27% change in emission intensity per 100 mV as opposed to ~10% seen in other common probes at comparable speeds7. In addition, wire-based PeT systems do not directly interact with the cellular electric field which produces minimal electrical interference and negligible changes in cellular capacitive load.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61890/61890fig01.jpg" /> 
Figure 1: Chemical, spectral and mechanistic parameters of VF2.1Cl dye.&#160;(A) Chemical structure of VF2.1Cl. Molecular features to note include multiple alkyl groups within the phenylene vinylene molecular wire which facilitate insertion into the plasma membrane. A negatively charged sulfonic acid group conjugated to the FITC probe ensures fluorophore stabilization on the extracellular surface and aids near perpendicular insertion relative to the electrical field of the lipid bilayer. (B) A simplified schematic of perpendicular VF2.1Cl embedding into the plasma membrane of a target cell. (C) Absorption and emission spectra of VF2.1Cl dye. Spectra is identical to that of standard FITC and GFP probes. (D) Depiction of the mechanistic mode of action of VF2.1Cl. In resting conditions (hyperpolarized), negative intracellular voltages drive free electrons towards the rostral fluorophore. Electron abundance ensures photo-induced electron transfer (PeT) is favored as a pathway out of the excited state after the optical excitation, effectively quenching fluorescence. In contrast, a depolarized membrane potential influences downward electron movement favoring fluorescence upon optical excitation. The resulting fluorescent response is linearly related to membrane voltage and can be precisely utilized to gather detailed temporal information on cellular electrophysiological kinetics. (E) Representative brightfield (upper) and fluorescence at 470 nm (lower) images of leporine cardiomyocytes loaded with VF2.1Cl. (F) Z stack of a single loaded cardiomyocyte. Arrows indicate areas of clear localization of VF2.1Cl to the cellular membrane. Images were acquired with a spinning disk confocal system consisting of a X-lightv3 spinning disk confocal head with a 50 &#181;m pinhole pattern; LDI-7 illuminator; Prime95B camera and a PlanApo Lambda 100x objective. Scale bar: 20 &#181;m. <a href="https://www.jove.com/files/ftp_upload/61890/61890fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The FITC probe conjugated to VF2.1Cl ensures that it can be used effectively under standard and GFP filter configurations, and it only requires a single channel acquisition system, both of which are common features of fluorescent imaging platforms. Analysis of dense human iPSC-CM monolayers with this dye has been recently reported8,9,10,11. Our protocol differs to these studies due to our investigation of single, isolated iPSC-CMs, unperturbed by the electrical and paracrine influences of dense syncytial monolayers, and our use of an affordable and customizable photometry system as opposed to complex confocal or wide-field imaging arrangements.
Here, we describe our protocol for the rapid acquisition and analysis of robust optical APs from isolated human iPSC-derived cardiomyocytes and native cardiomyocytes (see Supplementary File). We use VF2.1Cl coupled with a customizable state of the art platform for single cell photometry measurements.&#160;These experimental protocols have been approved by the ethics committee of the University Medical Center G&#246;ttingen (No. 10/9/15).

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	<tbody>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>0.25 Trypsin EDTA&#160;</td>
			<td>Gibco&#160;</td>
			<td>25200056</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 Supplement&#160;</td>
			<td>Gibco&#160;</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Carl Roth&#160;</td>
			<td>HN04.2</td>
			<td></td>
		</tr>
		<tr>
			<td>D(+)-Glucose anhydrous&#160;BioChemica</td>
			<td>ITW Reagents</td>
			<td>A1422</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum&#160;</td>
			<td>Gibco&#160;</td>
			<td>10270-106</td>
			<td></td>
		</tr>
		<tr>
			<td>FluoVolt Membrane Potential Kit&#160;</td>
			<td>Invitrogen&#160;</td>
			<td>F10488</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Carl Roth&#160;</td>
			<td>HN77.4</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma-Aldrich</td>
			<td>6781.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Lamanin</td>
			<td>Sigma-Aldrich</td>
			<td>114956-81-9</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel&#160;</td>
			<td>BD</td>
			<td>354230</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>9265.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Nifedipine&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>21829-25-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Invitrogen&#160;</td>
			<td>15140</td>
			<td></td>
		</tr>
		<tr>
			<td>ROCK Inhibitor Y27632</td>
			<td>Stemolecule</td>
			<td>04-0012-10</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 Medium&#160;</td>
			<td>Gibco&#160;</td>
			<td>61870010</td>
			<td></td>
		</tr>
		<tr>
			<td>Versene EDTA&#160;</td>
			<td>Gibco&#160;</td>
			<td>15040033</td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>495LP Dichroic Beamsplitter&#160;</td>
			<td>Chroma Technology</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Axopatch 200B Amplifier&#160;</td>
			<td>Molecular Devices</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Circle Coverslips, Thickness 0</td>
			<td>Thermo Scientific</td>
			<td>CB00100RA020MNT0</td>
			<td></td>
		</tr>
		<tr>
			<td>Digidata 1550B</td>
			<td>Molecular Devices</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dual OptoLED Power Supply&#160;</td>
			<td>Cairn Research&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ET470/40x Excitation Filter&#160;</td>
			<td>Chroma Technology</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ET535/50m</td>
			<td>Chroma Technology</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Etched Neubauer Hemacytometer</td>
			<td>Hausser Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Filter Cubes&#160;</td>
			<td>Cairn Research&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>IX73 Inverted Microscope&#160;</td>
			<td>Olympus&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MonoLED</td>
			<td>Cairn Research&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Multiport Adaptors&#160;</td>
			<td>Cairn Research&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Myopacer Cell Stimulator&#160;</td>
			<td>IonOptix</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Optomask Shutter&#160;</td>
			<td>Cairn Research&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Optoscan System Controller</td>
			<td>Cairn Research&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PH-1 Temperature Controlled Platform&#160;&#160;</td>
			<td>Warner Instruments&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Photomultiplier Detector&#160;</td>
			<td>Cairn Research&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PMT Amplifier Insert</td>
			<td>Cairn Research&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PMT Supply Insert</td>
			<td>Cairn Research&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>RC-26G Open Bath Chamber&#160;</td>
			<td>Warner Instruments&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SA-OLY/2AL Stage Adaptor&#160;</td>
			<td>Olympus&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>T565lpxr Dichroic Beamsplitter&#160;</td>
			<td>Chroma Technology</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>T660lpxr Dichroic Beamsplitter</td>
			<td>Chroma Technology</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TC-20 Dual Channel Temperature Controller&#160;</td>
			<td>npi Electronic</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>UPLFLN 40X Objective</td>
			<td>Olympus&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>USB 3.0 Colour Camera&#160;</td>
			<td>Imaging Source</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Clampex 11.1</td>
			<td>Molecular Devices&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Clampfit 11.1</td>
			<td>Molecular Devices&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>IC Capture 2.4&#160;</td>
			<td>Imaging Source&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Prism 8</td>
			<td>Graphpad</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

single-cell optical action potential measurement in human induced pluripotent stem cell-derived cardiomyocytes

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically carried out in low throughput. Rapid and ongoing expansion of induced pluripotent stem cell (iPSC) technology presents a new standard in cardiovascular research and alternate methods are now necessary to increase throughput of electrophysiological data at a single cell level. VF2.1Cl is a recently derived voltage sensitive dye which provides a rapid single channel, high magnitude response to fluctuations in membrane potential. It possesses kinetics superior to those of other existing voltage indicators and makes available functional data equivalent to that of traditional microelectrode techniques. Here, we demonstrate simplified, non-invasive action potential characterization in externally paced human iPSC derived cardiomyocytes using a modular and highly affordable photometry system.

<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61890/61890fig03.jpg" /> 
Figure 3: Optical action potential (AP) profiles of isolated native cardiomyocytes and human induced pluripotent stem cell derived cardiomyocytes (iPSC-CM).&#160;(A) Representative optical AP of a single murine cardiomyocyte (center) with Mean &#177; SEM of APD50 and APD90 (n = 7, right). (B) Representative optical AP of a singl...

Watch this Scientific Journal Video about Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes at JoVE.com

Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Here we describe optical acquisition and characterization of action potentials from induced pluripotent stem cell derived cardiomyocytes using a high-speed modular photometry system.

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically ...

This protocol can provide high throughput measurements of cellular electrophysiology in a manner that befits the rapidly developing technology of induced pluripotent stem cell-derived cardiomyocytes. The main advantage of this technique is that it is less complex and less labor-intensive than the traditional patch clamp technique, yet it is able to produce equivalent results. So this protocol can be expanded for large-scale toxicity screening programs using human induced pluripotent stem cell-derived cardiomyocytes.For example, the comprehensive in vitro proarrhythmia assay. To prepare induced pluripotent stem cell-derived cardiomyocyte cultures for the experiment, first coat individual 10 millimeter round glass number zero cover slips with 150 microliters of 1:60 basement membrane matrix within each well of a 24-well plate, and incubate the cover slips at four degrees Celsius for four hours. At the end of the incubation, remove the excess matrix from the cover slips and apply the appropriate volume of cells to each cover slip.After one hour at 37 degrees Celsius, carefully fill each well with plating medium and return the plate to the incubator. To prepare for voltage sensitive dye imaging, equip an inverted epifluorescence microscope with a 40x magnification high numerical aperture lens, and couple a fast switching warm white LED to the transmitted illumination port. Insert a simple red 660 nanometer filter into the transmitted light path and mount a fast switching 470 nanometer LED head for photometry recording.Insert a 470/40 excitation filter at the epifluorescence port to clean up the light generated by the LED, and insert a microscope cube containing a 495 nanometer long pass beam splitter in the mirror unit carousel within the microscope. Fit a detection arm containing an adjustable field diaphragm to the C mount port to allow region of interest selection, and couple a PMT detector and a USB camera to the microscope. Insert a filter cube containing a 565 nanometer long pass beam splitter and a 535/50 emission filter into the PMT port to split the emission light between the two detectors.Couple the PMT to a power supply and a PMT amplifier, and connect the PMT amplifier output to an analog input pin of a data acquisition system. To fulfill Nyquist criteria and to prevent aliasing, filter the analog data from the PMT at one kilohertz or higher, and digitize the data at a frequency that is at least double that of the highest frequency present in the analog signal. To load the cells with voltage sensitive dye, mix five microliters of 1000x FluoVolt and 50 microliters of power load solution, and add three microliters of the resulting loading solution to three milliliters of warmed Tyrode solution in a petri dish.Add a single induced pluripotent stem cell-derived cardiomyocyte coated cover slip to the dish and place the dish at 37 degrees Celsius for 20 minutes. Mount a heated live cell imaging chamber onto the microscope stage and fill the chamber with 500 microliters of fresh Tyrode solution. Then wash the cover slip with fresh Tyrode solution at 37 degrees Celsius and use fine point forceps to carefully transfer the cover slip to the pre-warmed bath chamber.To standardize the cellular dynamics and experimental parameters, insert two platinum electrodes into the chamber, spaced five millimeters apart, and connect the electrodes to an external stimulator. Set the stimulator to five millisecond bipolar field pulses at 0.5 hertz, and increase the stimulus from one volt until the cells begin to contract. Then set the voltage to roughly 25%above this threshold.For optical action potential acquisition, use the transmitted light path and the USB camera to visualize the myocytes under brightfield conditions. Select an isolated cell and use the field diaphragm to tightly crop its optical path, ensuring that only light from the cell of interest is monitored. Activate the PMT amplifier, and set the PMT supply to 750 volts.Run the stimulation protocol along with the acquisition software while simultaneously activating the 470 nanometer excitation light. Record 10 sweeps, ensuring that stable action potentials are detected. After the last sweep, immediately move the microscope stage while still recording to briefly acquire background signal from a region devoid of cells and turn off the excitation light.To analyze the action potential data, open a saved recording in the appropriate analysis software and average 10 sweeps containing stimulated action potentials from a single cell. Subtract the mean of the baseline signal representing fluorescence offset from the average trace, then use the formula to calculate the change in fluorescence using diastolic fluorescence as F0.Identify the diastolic and action potential areas of interest and measure the desired cardiac action potential parameters, including, but not limited to, the duration at 50%and 90%repolarization. Then export the data from this single cell to a spreadsheet program.This method allows for rapid and precise quantification of repolarization mechanics, which can provide valuable insights into cellular ionic abnormalities. Pronounced phase one features can be observed in optical signals from murine cardiomyocytes, which are morphologically distinct from those of human induced pluripotent stem cell-derived cardiomyocytes. In addition, human induced pluripotent stem cell-derived cardiomyocytes are responsive to pharmacological manipulation with nifedipine, a known L-type calcium channel antagonist.During continuous drug application, a 41.5%decrease in action potential duration at 90%repolarization was observed, suggesting the functionality of fluorescent voltage indicator based imaging as a platform for prospective high throughput cardiac drug screening studies. It is important to keep in mind that although the voltage sensitive dye used in this study is less cytotoxic than others, the excitation light should still be used sparingly to conserve cellular viability. The modulatory photometry equipment used in this protocol is incredibly versatile.For example, it can be used for simultaneous measurements of other electrophysiological parameters, such as intracellular calcium.

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