This work was supported by the Slovenian Research and Innovation Agency (ARIS) within research program P2-0249, infrastructure program I0-0022, and research project J2-2503. The work was in part supported by ARIS and the University of Ljubljana through funding for Start-up Research Programs. The work was in part supported by the European Union and ARIS through NextGenerationEU and NOO funding within project MN-0023. The work was in part supported by the European Union through the ERC Starting Grant (No. 101115323 - REINCARNATION) and Marie Sk&#322;odowska-Curie fellowship (No. 893077 - EPmIC). Views and opinions expressed are, however, those of the author(s) only and do not necessarily reflect those of the European Union or the European Research Council. Neither the European Union nor the granting authority can be held responsible for them. The authors thank the group of Adam E. Cohen from Harvard University for kindly providing the tet-on spiking HEK cells and Du&#353;a Hod&#382;i&#263; for her help in the cell culture laboratory.

1. Preparation of reagents and cell culture
<ol>
	<li>Prepare the cell culture medium by supplementing Dulbecco&#39;s Modified Eagle&#39;s Medium-high glucose with 10% fetal bovine serum, 2 mM glutamine, penicillin-streptomycin (penicillin 100 U/mL, streptomycin 100 &#181;g/mL), 2 &#181;g/mL puromycin, 5 &#181;g/mL blasticidin, and 200 &#181;g/ mL geneticin.</li>
	<li>Prepare phosphate-buffered saline (PBS) solution with 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 1.8 mM KH2PO4, pH 7.4. Filter it through a sterilizing membrane filter with a 0.22 &#181;m pore size.</li>
	<li>Prepare Tyrode solution with 2 mM KCl, 125 mM NaCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, 30 mM glucose, pH 7.3. Prepare low potassium Tyrode solution with 0.5 mM KCl, 126.5 mM NaCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, 30 mM glucose, pH 7.3. Filter both solutions through a sterilizing membrane filter with a 0.22 &#181;m pore size.</li>
	<li>Prepare 1 mg/mL poly-L-lysine stock solution by adding 5 mL of PBS in a vial containing 5 mg of poly-L-lysine.</li>
	<li>Prepare 2 mg/mL doxycycline stock solution in distilled water and filter it through a sterilizing membrane filter with a 0.22 &#181;m pore size.</li>
	<li>Prepare 4 mM potentiometric dye ElectroFluor630 stock solution in pure ethanol (add 25 &#181;L of pure ethanol to the tube containing 100 nM - 73 &#181;g of the dye).</li>
	<li>Culture genetically engineered HEK cells in a T25 flask in 6 mL of the cell culture medium as previously described8. The ingredients of the culture medium are light sensitive; thus, keep the medium protected from light (wrap the medium flask in aluminum foil) and perform passaging in low light conditions.</li>
	<li>Passage the cells every 3-4 days by seeding 3 x 105 or 5 x 105 cells to a fresh T25 flask. Count the cells using a hemocytometer. Maintain cells in a master flask as non-excitable NS-HEK cells without doxycycline addition because they do not tolerate sustained Kir2.1 expression. Use cells from passages lower than 14 in the experiments.</li>
</ol>
2. Sample preparation
<ol>
	<li>Poly-L-lysine-coating of cover glass chamber slides
	<ol>
		<li>Pipette 1 mL of PBS to one well of the 2-well cover glass chamber slides, add 10 &#181;L of sterile 1 mg/mL poly-L-lysine solution and mix well with a pipette.</li>
		<li>Incubate for 1.5 h at room temperature in sterile conditions in a laminar flow hood. Remove the PBS with poly-L-lysine without any further washing.</li>
	</ol>
	</li>
	<li>Seeding the cells
	<ol>
		<li>Prepare a 1:10 dilution of doxycycline stock solution in a culture medium in a 1.5 mL tube. For each well 30 &#181;L of 1:10 doxycycline dilution is needed (3 &#181;L of doxycycline stock solution and 27 &#181;L of culture medium), prepare the final volume depending on the total number of experimental wells.</li>
		<li>Remove the culture medium from the T25 flask and trypsinize the cells with 2.5 mL of Trypsin-EDTA solution in a CO2 incubator at 37 &#176;C for 2 min.</li>
		<li>Add 2.5 mL of fresh culture medium and, by gentle pipetting, detach the cells from the surface. Mix the cell suspension well.</li>
		<li>Count the cells with a hemocytometer and determine the density of cells in suspension.</li>
		<li>Calculate the volume of trypsinized cell suspension needed to establish the culture. For 2-day culture, seed 2.5 x 105 cells, and for 3-day culture, seed 1 x 105 cells per well. Adjust the exact number of cells if needed.</li>
		<li>Calculate the volume of the culture medium by subtracting the previously calculated cell suspension volume from 1470 &#181;L. The remaining 30 &#181;L represents the volume of 1:10 doxycycline dilution. The total volume in each well will be 1500 &#181;L (cells, doxycycline, and culture medium).</li>
		<li>For preparing samples with excitable S-HEK cells, pipette the calculated volume of culture medium and trypsinized cell suspension into one well and add 30 &#181;L of 1:10 doxycycline dilution. Use only one well of the two-well chambers since the experiment takes a long time (around 40 min). Mix well by gentle pipetting just before placing the chambers into the CO2 incubator.</li>
		<li>For preparing samples with non-excitable NS-HEK cells, pipette the calculated volume of culture medium into a well and add 90% of the calculated volume of cell suspension for S-HEK cells without doxycycline. NS-HEK cells grow slightly faster than S-HEK cells.</li>
		<li>Incubate the chambers in a 5% CO2 incubator at 37 &#176;C for 2-3 days prior to the experiment. For samples with doxycycline, this incubation time is sufficient to render the cells excitable.</li>
	</ol>
	</li>
	<li>Labeling the cells
	<ol>
		<li>Before the experiment, pipette 3 &#181;L of potentiometric dye stock solution to a 1.5 mL tube and leave the open tube at room temperature in a laminar flow hood to allow the ethanol to evaporate and the potentiometric dye to dry (approximately 15 min). Add 997 &#181;L of cold culture medium (from a refrigerator at 4 &#176;C) to the tube and dissolve the dye to a final concentration of 12 &#181;M.</li>
		<li>Remove the culture medium from the well and add 1 mL of potentiometric dye solution to the cells. Refrigerate them at 4 &#176;C and incubate for 20 min.</li>
		<li>Remove the potentiometric dye solution and save it in the tube for further reuse (for up to six sequential experiments in 1 day).</li>
		<li>Gently wash the cells 3x with Tyrode solution, and after the last wash, pipette 1 mL of low potassium Tyrode solution into the well.</li>
	</ol>
	</li>
</ol>
3. Electrostimulation and electroporation
<ol>
	<li>Configure synchronization of the pulse delivery with image acquisition using a TTL signal from the microscope system that triggers the pulse generator. For generation of high-voltage pulses, use a custom pulse generator11 or any pulse generator that can be triggered externally with a TTL signal. 
	NOTE: In the presented experimental configuration, LAS X software is used for image acquisition, which can be configured to send a TTL signal at a preselected time during a time-lapse acquisition.</li>
	<li>Place two parallel Pt/Ir wire electrodes, with 0.8 mm diameter and 5 mm distance between inner edges, at the bottom of the chamber and fix the chamber under the microscope.</li>
	<li>Connect the electrodes to the pulse generator. Connect the output of the pulse generator to an oscilloscope using a voltage and a current probe to enable verification that the pulses were correctly delivered during the experiment.</li>
	<li>Focus the cells in brightfield. Image the cells that are located roughly in the middle between the electrodes.</li>
	<li>Start the first acquisition of fluorescence images in time-lapse mode with a 40x objective, recording the fluorescence signal in the whole field of view. The camera should be fast enough to acquire a clear time course of an action potential (ideally higher than 25 frames/s).
	<ol>
		<li>Acquire 80 images in a time-lapse mode, with at least 25 frames/s to resolve the real-time course of the action potential. The total duration of image acquisition results in around 3&#160;s. During image acquisition, illuminate the cells with an excitation wavelength of 635 nm, exposure time of 10 ms, and detect the emission at around 700 nm. In this first acquisition, do not apply any pulse - this acquisition will be used to correct the subsequent acquisitions for dye photobleaching. After this acquisition, wait for 2 min. 
		NOTE: This experiment uses 27.8 frames/s, i.e., 36 ms period, which is the time from the beginning of the first image acquisition to the beginning of the next image acquisition in the time-lapse recording).</li>
	</ol>
	</li>
	<li>Every 2 min, record a time-lapse in the same way as in step 3.5. For these acquisitions, apply a single 100 &#181;s pulse at the time of acquisition of the 10th image (at 324 ms). Acquire images with the following pulse voltages: 63 V, 75 V, 88 V, 100 V, 125 V, 150 V, 175 V, and 200 V. Estimate the electric field to which the cells are exposed as the applied voltage-to-electrode-distance ratio (E = 126, 150, 176, 200, 250, 300, 350, and 400 V/cm). 
	NOTE: At lower electric fields, the pulses will trigger action potentials; at higher electric fields, they will trigger electroporation, which manifests as a prolonged depolarization. The duration of the experiment on each sample of cells is approximately 40 min (20 min for labeling and 20 min for microscopy).</li>
</ol>
4. Data analysis
<ol>
	<li>Export the time-lapse recordings to tiff format.</li>
	<li>Place time-lapse recordings of all the applied voltages during an individual experiment (one sample of cells) into a single folder. Rename the time-lapse recordings in such a form that electric field E can be recognized by the MATLAB application: Vcm (e.g., 126Vcm for 126 V/cm). The remaining parts of the name can be arbitrary. For the acquisition at the beginning of the experiment where no pulse is applied, rename using the phrase 000Vcm. An example of the time-lapse recording names: 2022_1_20 1DOX 000Vcm, 2022_1_20 1DOX 126Vcm, 2022_1_20 1DOX 150Vcm, etc.</li>
	<li>Open the application, which can be downloaded from 
	https://github.com/bor-kos/fluorescenceActionPotential. Click on&#160;Select Data Folder to choose the folder containing time-lapse recordings of one experiment.</li>
	<li>Select the pixels to be used for further data analysis. To do this, use two sliders to determine the low threshold for a clear image of the membranes (in purple) and the high threshold for eliminating debris (large spots with high fluorescence) from the analysis (in green).</li>
	<li>Select Analyze Data. In the Analysis tab, a table with extracted parameters appears. On the right side of the table, graphs of relative fluorescence in time appear for each E used. Select graphs for a given E from the drop-down menu on the left side above the table.</li>
	<li>Examine all the graphs and perform manual correction in the case of false peak detection. Select Clear AUTO Peaks at the bottom in the Analysis tab, and the peaks that were detected automatically are cleared. Click on the Graph to manually determine a new peak time point.</li>
	<li>Select Rerun AUTO Peak Detection at the bottom in the Analysis tab to perform automatic peak detection again. The extracted parameters will now be adjusted to the new peak.</li>
	<li>Select Export Data at the bottom right in the Analysis tab to export data to the same folder in the form of a .mat file, spreadsheet file, and PNG images for each E used.</li>
</ol>

Evaluating Electroporation Effects on Genetically Engineered Spiking HEK Cells

<ol>
	<li>Kotnik, T., Rems, L., Tarek, M., Miklav&#269;i&#269;, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+electroporation+and+electropermeabilization:+Mechanisms+and+models.">Membrane electroporation and electropermeabilization: Mechanisms and models.</a> Ann Rev Biophys. 48 (1), 63-91 (2019).</li><li>Yarmush, M. L., Golberg, A., Ser&#353;a, G., Kotnik, T., Miklav&#269;i&#269;, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electroporation-based+technologies+for+medicine:+principles,+applications,+and+challenges.">Electroporation-based technologies for medicine: principles, applications, and challenges.</a> Ann Rev Biomed Eng. 16, 295-320 (2014).</li><li>Kotnik, T., 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=Electroporation-based+applications+in+biotechnology.">Electroporation-based applications in biotechnology.</a> Trend Biotechnol. 33 (8), 480-488 (2015).</li><li>Mahni&#269;-Kalamiza, S., Vorobiev, E., Miklav&#269;i&#269;, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electroporation+in+food+processing+and+biorefinery.">Electroporation in food processing and biorefinery.</a> J Memb Biol. 247 (12), 1279-1304 (2014).</li><li>Reddy, V. Y., 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=Pulsed+field+or+conventional+thermal+ablation+for+paroxysmal+atrial+fibrillation.">Pulsed field or conventional thermal ablation for paroxysmal atrial fibrillation.</a> New Engl J Med. 389 (18), 1660-1671 (2023).</li><li>Cvetkoska, A., Ma&#269;ek-Lebar, A., Trdina, P., Miklav&#269;i&#269;, D., Reber&#353;ek, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscle+contractions+and+pain+sensation+accompanying+high-frequency+electroporation+pulses.">Muscle contractions and pain sensation accompanying high-frequency electroporation pulses.</a> Sci Rep. 12 (1), 8019 (2022).</li><li>Tung, L., Tovar, O., Neunlist, M., Jain, S. K., O'neill, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+strong+electrical+shock+on+cardiac+muscle+tissue.">Effects of strong electrical shock on cardiac muscle tissue.</a> Ann New York Acad Sci. 720 (1), 160-175 (1994).</li><li>Batista Napotnik, T., 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=Genetically+engineered+HEK+cells+as+a+valuable+tool+for+studying+electroporation+in+excitable+cells.">Genetically engineered HEK cells as a valuable tool for studying electroporation in excitable cells.</a> Sci Rep. 14 (1), 720 (2024).</li><li>McNamara, H. M., Zhang, H., Werley, C. A., Cohen, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optically+controlled+oscillators+in+an+engineered+bioelectric+tissue.">Optically controlled oscillators in an engineered bioelectric tissue.</a> Phys Rev X. 6 (3), 031001 (2016).</li><li>Tian, H., 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=Video-based+pooled+screening+yields+improved+far-red+genetically+encoded+voltage+indicators.">Video-based pooled screening yields improved far-red genetically encoded voltage indicators.</a> Nat Methods. 20 (7), 1082-1094 (2023).</li><li>Sweeney, D. C., 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=Quantification+of+cell+membrane+permeability+induced+by+monopolar+and+high-frequency+bipolar+bursts+of+electrical+pulses.">Quantification of cell membrane permeability induced by monopolar and high-frequency bipolar bursts of electrical pulses.</a> Biochim Biophys Acta Biomembranes. 1858 (11), 2689-2698 (2016).</li><li>Pakhomov, A. G., 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=Long-lasting+plasma+membrane+permeabilization+in+mammalian+cells+by+nanosecond+pulsed+electric+field+(nsPEF).">Long-lasting plasma membrane permeabilization in mammalian cells by nanosecond pulsed electric field (nsPEF).</a> Bioelectromagnetics. 28 (8), 655-663 (2007).</li><li>Lee, 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=In+vivo+ratiometric+optical+mapping+enables+high-resolution+cardiac+electrophysiology+in+pig+models.">In vivo ratiometric optical mapping enables high-resolution cardiac electrophysiology in pig models.</a> Cardiovasc Res. 115 (11), 1659-1671 (2019).</li></ol>

The authors declare no conflict of interest.

S-HEK cells are a simple excitable cell model with well-defined sodium and potassium channels, NaV1.5 and Kir2.19. This enables us to perform in-depth studies of how excitable cells respond to applied electric pulses and link the experimental data with mathematical models based on the well-known theoretical characteristics of the channels expressed in S-HEK cells. In our previous study8, we described the responses of these cells to 100 &#181;s pulses o...

In electroporation, high-voltage electric pulses cause an increase in plasma membrane permeability for molecules that are otherwise poorly permeant1. Electroporation-based techniques are nowadays widely used in applications in medicine2, biotechnology3, and food technology4.
Excitable cells such as&#160;neuronal and muscle cells are among the primary targets in rapidly emerging electroporation-based treatments in the heart, brain, and skeletal muscles5. Moreover, they can be affected by electric pulses even in therapies where they are not the primary targets, and this can cause adverse side effects such as pain, muscle twitching, and nerve damage6. Namely, electric pulses can cause electrical stimulation of excitable cells: they activate voltage-gated ion channels and trigger action potentials (AP). However, at higher electric fields, the cells&#39; plasma membrane becomes permeabilized, and additional ionic current through pores/defects occurs7. This additional ionic current affects cell excitability in a complex interplay between excitation and electroporation. Therefore, to optimize the electroporation-based treatments of excitable and non-excitable tissues, there is a need to study the effects of electric pulses on excitable cells, their ion channels, and excitability in vitro.
In a recent study8, we explored genetically engineered human embryonic kidney (HEK) cells developed by Cohen et al.9,10, as a valuable tool for studying electroporation in excitable cells. In these otherwise non-excitable cells, a minimal complement of sodium and potassium channels (NaV1.5 and Kir2.1) needed for excitability is expressed. The expression of Kir2.1 in these cells is controlled by a doxycycline-induced tet-on system that allows the establishment of two cell variants: excitable spiking S-HEK (containing NaV1.5 and Kir2.1) and non-excitable non-spiking NS-HEK cells (containing only NaV1.5 channels). This allows us to compare the effects of electric pulses on excitable and non-excitable variants of the same type of cells. Compared to isolated primary excitable cells, excitable cells differentiated from embryonic or induced pluripotent stem cells, and cell lines derived from excitable tissues, S-HEK cells have several advantages: they are easy to handle, cultivate and propagate, have well-defined sodium and potassium channels, and generate robust APs8.
We designed a protocol for monitoring changes in transmembrane voltage (TMV) triggered by electric pulses of different amplitudes with the use of a fluorescent potentiometric dye. This way, we can study how electroporation affects AP generation in S-HEK cells and other changes in the TMV in both S-HEK and NS-HEK cells. Briefly, the S-HEK or NS-HEK cells are exposed to a single 100 &#181;s electric pulse every 2 min, and relative changes in fluorescence are recorded for roughly 3 s around each pulse application. The applied pulses are of increasing voltage, first triggering APs&#160;in cells and, at a higher electric field, electroporation. The 2 min interval between pulse application was chosen to minimize the possible cumulative effects of consecutive pulses while keeping experiments short enough not to deteriorate the cells. The image processing was automated with a custom MATLAB application to extract relevant parameters of the fluorescence responses of the potentiometric dye, hence evaluating the changes in TMV (APs and&#160;sustained depolarization due to electroporation) in S-HEK and NS-HEK&#160;cells.

<table><tbody><tr><td>1.5 mL tube</td><td>Ratiolab&amp;nbsp;</td><td>5615000</td><td></td></tr><tr><td>2-well Nunc Lab-Tek II Chambered #1.5 German Coverglass System</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>155379</td><td></td></tr><tr><td>ADP305 Differential voltage probe</td><td>Teledyne LeCroy</td><td></td><td></td></tr><tr><td>AP015 Current probe</td><td>Teledyne LeCroy</td><td></td><td></td></tr><tr><td>Blasticidin&amp;nbsp;</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>A1113903</td><td></td></tr><tr><td>CO2 incubator&amp;nbsp;</td><td>PHCbi&amp;nbsp;</td><td>MCO -230AICUV</td><td></td></tr><tr><td>Doxycycline&amp;nbsp;</td><td>Sigma-Aldrich/Merck</td><td>D9891</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s Modifed Eagle&amp;rsquo;s Medium high glucose growth medium</td><td>Sigma-Aldrich/Merck</td><td>D5671</td><td></td></tr><tr><td>ElectroFluor630 (Di-4-ANEQ(F)PTEA)</td><td>Potentiometric Probes</td><td>31795</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Sigma-Aldrich/Merck</td><td>F2442</td><td></td></tr><tr><td>Geneticin</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>10131035</td><td></td></tr><tr><td>Glutamine</td><td>Sigma-Aldrich/Merck</td><td>G7513</td><td></td></tr><tr><td>Laminar flow hood</td><td>Iskra Pio&amp;nbsp;</td><td>MC 15-2</td><td></td></tr><tr><td>MATLAB</td><td>Mathworks</td><td></td><td></td></tr><tr><td>Penicillin&amp;ndash;streptomycin</td><td>Sigma-Aldrich/Merck</td><td>P07681</td><td></td></tr><tr><td>Poly-L-lysine hydrobromide&amp;nbsp;</td><td>Sigma-Aldrich/Merck</td><td>P9155-5MG&amp;nbsp;&amp;nbsp;</td><td></td></tr><tr><td>Pulse generator&amp;nbsp;</td><td>Custom made</td><td></td><td>see reference&lt;sup&gt;11&lt;/sup&gt;</td></tr><tr><td>Puromycin</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>A1113803</td><td></td></tr><tr><td>T25 25cm&amp;sup2; Tissue culture flask</td><td>TPP&amp;nbsp;</td><td>90026</td><td></td></tr><tr><td>Tet-on spiking HEK cells</td><td>American Type Culture Collection ATCC</td><td>CRL-3479</td><td></td></tr><tr><td>Thunder Imager Live Cell system for fluorescence microscopy</td><td>Leica Microsystems&amp;nbsp;</td><td></td><td></td></tr><tr><td>Trypsin-EDTA solution</td><td>Sigma-Aldrich/Merck</td><td>T3924</td><td></td></tr><tr><td>WavePro 7300A Oscilloscope</td><td>Teledyne LeCroy</td><td></td><td></td></tr></tbody></table>

monitoring electroporation-induced changes in action potential generation in genetically engineered tet-on spiking hek cells

Excitable cells such as&#160;neuronal and muscle cells can be primary targets in rapidly emerging electroporation-based treatments. However, they can be affected by electric pulses even in therapies where they are not the primary targets, and this can cause adverse side effects. Therefore, to optimize the electroporation-based treatments of excitable and non-excitable tissues, there is a need to study the effects of electric pulses on excitable cells, their ion channels, and excitability in vitro. For this purpose, a protocol was developed for optical monitoring of changes in action potential generation due to electroporation on a simple excitable cell model of genetically engineered tet-on spiking HEK cells. With the use of a fluorescent potentiometric dye, the changes in transmembrane voltage were monitored under a fluorescence microscope, and relevant parameters of cell responses were extracted automatically with a MATLAB application. This way, the excitable cell responses to different electric pulses and the interplay between excitation and electroporation could&#160;be efficiently evaluated.

Changes in TMV in excitable cells in vitro triggered by electric pulses can be optically monitored with this protocol, and the&#160;fluorescence signals can be analyzed to extract their relevant parameters.
The experimental setup is shown in Figure 1A. A typical signal of 100 &#181;s electric pulse used in experiments is shown in Figure 1B. Cells in the chambers that are placed under the microscope typically look like the cel...

Monitoring Electroporation-Induced Changes in Action Potential Generation in Genetically Engineered Tet-On Spiking HEK cells

For studying responses of excitable cells in vitro, the protocol describes optical monitoring of changes in action potential generation due to electroporation on a simple excitable cell model of genetically engineered tet-on spiking HEK cells as well as changes in transmembrane voltage with automated extraction of relevant parameters.

Excitable cells such as&#160;neuronal and muscle cells can be primary targets in rapidly emerging electroporation-based treatments. However, they can be ...

monitoring-electroporation-induced-changes-action-potential

Research

JoVE Journal

Bioengineering

233 Views.  University of Ljubljana. Elektroporatie verhoogt tijdelijk de permeabiliteit van het celmembraan door middel van elektrische pulsen met hoge spanning. Er zijn verschillende zich snel ontwikkelende klinische behandelingen op basis van elektroporatie die gericht zijn op spier- en neuronale cellen. Het is dus van cruciaal belang om te onderzoeken hoe elektroporatie het vermogen van deze exciteerbare cellen om actiepotentialen te activeren beïnvloedt.In-vitro-experimenten zijn er...

Video: Monitoring van door elektroporatie geïnduceerde veranderingen in het genereren van actiepotentieel in genetisch gemanipuleerde Tet-On Spiking HEK-cellen

Single Cell Electroporation in vivo within the Intact Developing Brain

Single-cell electroporation (SCE) is a specialized technique allowing the delivery of DNA or other macromolecules into individual cells within intact tissue, including in vivo preparations. The distinct advantage of this technique is that experimental manipulations may be performed on individual cells while leaving the surrounding tissue unaltered, thereby distinguishing cell-autonomous effects from those resulting from global treatments. When combined with advanced in vivo imaging techniques, SCE of fluorescent markers permits direct visualization of cellular morphology, cell growth, and intracellular events over timescales ranging from seconds to days. While this technique is used in a variety of in vivo and ex vivo preparations, we have optimized this technique for use in Xenopus laevis tadpoles. In this video article, we detail the procedure for SCE of a fluorescent dye or plasmid DNA into neurons within the intact brain of the albino Xenopus tadpole. We also discuss methods to optimize yield, and show examples of live two-photon fluorescence imaging of neurons fluorescently labeled by SCE.

Single-cell electroporation (SCE) is a specialized technique allowing delivery of DNA or other macromolecules into individual cells within intact tissue, including in vivo preparations. Here we detail the procedure for SCE of a fluorescent dye or plasmid DNA into neurons within the intact brain of the Xenopus laevis tadpole.

Single-cell electroporation (SCE) is a specialized technique allowing the delivery of DNA or other macromolecules into individual cells within intact ...

Biology

Measuring the Induced Membrane Voltage with Di-8-ANEPPS

Placement of a cell into an external electric field causes a local charge redistribution inside and outside of the cell in the vicinity of the cell membrane, resulting in a voltage across the membrane. This voltage, termed the induced membrane voltage (also induced transmembrane voltage, or induced transmembrane potential difference) and denoted by &Delta;&Phi;, exists only as long as the external field is present. If the resting voltage is present on the membrane, the induced voltage superimposes (adds) onto it. By using one of the potentiometric fluorescent dyes, such as di-8-ANEPPS, it is possible to observe the variations of &Delta;&Phi; on the cell membrane and to measure its value noninvasively. di-8-ANEPPS becomes strongly fluorescent when bound to the lipid bilayer of the cell membrane, with the change of the fluorescence intensity proportional to the change of &Delta;&Phi;. This video shows the protocol for measuring &Delta;&Phi; using di-8-ANEPPS and also demonstrates the influence of cell shape on the amplitude and spatial distribution of &Delta;&Phi;.

External electric field induces a voltage on the membrane of a cell, termed the induced membrane voltage (&Delta;&Phi;). By using the potentiometric dye di-8-ANEPPS, it is possible to measure the &Delta;&Phi; noninvasively. This video shows the protocol for measuring &Delta;&Phi; using di-8-ANEPPS.

Placement of a cell into an external electric field causes a local charge redistribution inside and outside of the cell in the vicinity of the cell ...

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

Optimized Transfection Strategy for Expression and Electrophysiological Recording of Recombinant Voltage-Gated Ion Channels in HEK-293T Cells

The in vitro expression and electrophysiological recording of recombinant voltage-gated ion channels in cultured human embryonic kidney cells (HEK-293T) is a ubiquitous research strategy. HEK-293T cells must be plated onto glass coverslips at low enough density so that they are not in contact with each other in order to allow for electrophysiological recording without confounding effects due to contact with adjacent cells. Transfected channels must also express with high efficiency at the plasma membrane for whole-cell patch clamp recording of detectable currents above noise levels. Heterologous ion channels often require long incubation periods at 28&deg;C after transfection in order to achieve adequate membrane expression, but there are increasing losses of cell-coverslip adhesion and membrane stability at this temperature. To circumvent this problem, we developed an optimized strategy to transfect and plate HEK-293T cells. This method requires that cells be transfected at a relatively high confluency, and incubated at 28&deg;C for varying incubation periods post-transfection to allow for adequate ion channel protein expression. Transfected cells are then plated onto glass coverslips and incubated at 37&deg;C for several hours, which allows for rigid cell attachment to the coverslips and membrane restabilization. Cells can be recorded shortly after plating, or can be transferred to 28&deg;C for further incubation. We find that the initial incubation at 28&deg;C, after transfection but before plating, is key for the efficient expression of heterologous ion channels that normally do not express well at the plasma membrane. Positively transfected, cultured cells are identified by co-expressed eGFP or eGFP expressed from a bicistronic vector (e.g. pIRES2-EGFP) containing the recombinant ion channel cDNA just upstream of an internal ribosome entry site and an eGFP coding sequence. Whole-cell patch clamp recording requires specialized equipment, plus the crafting of polished recording electrodes and L-shaped ground electrodes from borosilicate glass. Drug delivery to study the pharmacology of ion channels can be achieved by directly micropipetting drugs into the recording dish, or by using microperfusion or gravity flow systems that produce uninterrupted streams of drug solution over recorded cells.

Reliable method for highly efficient in vitro expression and subsequent electrophysiological recording of recombinant voltage-gated ion channels in cultured human embryonic kidney cells (HEK-293T).

The in vitro expression and electrophysiological recording of recombinant voltage-gated ion channels in cultured human embryonic kidney cells (HEK-293T) ...

Imaging Membrane Potential with Two Types of Genetically Encoded Fluorescent Voltage Sensors

Genetically encoded voltage indicators (GEVIs) have improved to the point where they are beginning to be useful for in vivo recordings. While the ultimate goal is to image neuronal activity in vivo, one must be able to image activity of a single cell to ensure successful in vivo preparations. This procedure will describe how to image membrane potential in a single cell to provide a foundation to eventually image in vivo. Here we describe methods for imaging GEVIs consisting of a voltage-sensing domain fused to either a single fluorescent protein (FP) or two fluorescent proteins capable of F&#246;rster resonance energy transfer (FRET) in vitro. Using an image splitter enables the projection of images created by two different wavelengths onto the same charge-coupled device (CCD) camera simultaneously. The image splitter positions a second filter cube in the light path. This second filter cube consists of a dichroic and two emission filters to separate the donor and acceptor fluorescent wavelengths depending on the FPs of the GEVI. This setup enables the simultaneous recording of both the acceptor and donor fluorescent partners while the membrane potential is manipulated via whole cell patch clamp configuration. When using a GEVI consisting of a single FP, the second filter cube can be removed allowing the mirrors in the image splitter to project a single image onto the CCD camera.

A method for imaging changes in membrane potential using genetically encoded voltage indicators is described.

Genetically encoded voltage indicators (GEVIs) have improved to the point where they are beginning to be useful for in vivo recordings. While the ultimate ...

Neuroscience

The Fabrication and Operation of a Continuous Flow, Micro-Electroporation System with Permeabilization Detection

Current therapeutic innovations, such as CAR-T cell therapy, are heavily reliant on viral-mediated gene delivery. Although efficient, this technique is accompanied by high manufacturing costs, which has brought about an interest in using alternative methods for gene delivery. Electroporation is an electro-physical, non-viral approach for the intracellular delivery of genes and other exogenous materials. Upon the application of an electric field, the cell membrane temporarily allows molecular delivery into the cell. Typically, electroporation is performed on the macroscale to process large numbers of cells. However, this approach requires extensive empirical protocol development, which is costly when working with primary and difficult-to-transfect cell types. Lengthy protocol development, coupled with the requirement of large voltages to achieve sufficient electric-field strengths to permeabilize the cells, has led to the development of micro-scale electroporation devices. These micro-electroporation devices are manufactured using common microfabrication techniques and allow for greater experimental control with the potential to maintain high throughput capabilities. This work builds off a microfluidic-electroporation technology capable of detecting the level of cell membrane permeabilization at a single-cell level under continuous flow. However, this technology was limited to 4 cells processed per second, and thus a new approach for increasing the system throughput is proposed and presented here. This new technique, denoted as cell-population-based feedback control, considers the cell permeabilization response to a variety of electroporation pulsing conditions and determines the best-suited electroporation pulse conditions for the cell type under test. A higher-throughput mode is then used, where this 'optimal' pulse is applied to the cell suspension in transit. The steps for fabricating the device, setting up and running the microfluidic experiments, and analyzing the results are presented in detail. Finally, this micro-electroporation technology is demonstrated by delivering a DNA plasmid encoding for green fluorescent protein (GFP) into HEK293 cells.

This protocol describes the microfabrication techniques required to build a lab-on-a-chip, microfluidic electroporation device. The experimental setup performs controlled, single-cell-level transfections in a continuous flow and can be extended to higher throughputs with population-based control. An analysis is provided showcasing the ability to electrically monitor the degree of cell membrane permeabilization in real-time.

Current therapeutic innovations, such as CAR-T cell therapy, are heavily reliant on viral-mediated gene delivery. Although efficient, this technique is ...

TEEI Method for Electroporation Analysis and Delivery Prediction in A431 Cells

Porous Substrate-Based Electroporation with Transepithelial Electrical Impedance Monitoring

Porous substrate electroporation (PSEP) is an emerging method of electroporation that provides high throughput and consistent delivery. Like many other types of intracellular delivery, PSEP relies heavily on fluorescent markers and fluorescent microscopy to determine successful delivery. To gain insight into the intermediate steps of the electroporation process, a PSEP platform with integrated transepithelial electrical impedance (TEEI) monitoring was developed. Cells are cultured in commercially available inserts with porous membranes. After a 12 h incubation period to allow for the formation of a fully confluent cell monolayer, the inserts are placed in transfection media located in the wells of the PSEP device. The cell monolayers are then subjected to a user-defined waveform, and delivery efficiency is confirmed through fluorescent microscopy. This workflow can be significantly enhanced with TEEI measurements between pulsing and fluorescent microscopy to collect additional data on the PSEP process, and this additional TEEI data is correlated with delivery metrics such as delivery efficiency and viability. This article describes a protocol for performing PSEP with TEEI measurements.

Author Spotlight: Optimizing Porous Substrate Electroporation Through Micro and Nanochannels for Enhanced Monitoring and Intermediate Stage Characterization

Porous substrate electroporation (PSEP) pairs consistent, high throughput delivery with high cell viability. Introduction of transepithelial electrical impedance (TEEI) measurements provides insight into the intermediate processes of PSEP and allows for label-free delivery. This article discusses a method for performing PSEP delivery experiments and TEEI measurement analysis simultaneously.

Porous substrate electroporation (PSEP) is an emerging method of electroporation that provides high throughput and consistent delivery. Like many other ...

A Functional Assay for Gap Junctional Examination; Electroporation of Adherent Cells on Indium-Tin Oxide

In this technique, cells are cultured on a glass slide that is partly coated with indium-tin oxide (ITO), a transparent, electrically conductive material. A variety of molecules, such as peptides or oligonucleotides can be introduced into essentially 100% of the cells in a non-traumatic manner.&#160; Here, we describe how it can be used to study intercellular, gap junctional communication. Lucifer yellow penetrates into the cells when an electric pulse, applied to the conductive surface on which they are growing, causes pores to form through the cell membrane. This is electroporation. Cells growing on the nonconductive&#160;glass surface immediately adjacent to the electroporated region do not take up Lucifer yellow by electroporation but do acquire the fluorescent dye as it is passed to them via gap junctions that link them to the electroporated cells. The results of the transfer of dye from cell to cell can be observed microscopically under fluorescence illumination. This technique allows for precise quantitation of gap junctional communication. In addition, it can be used for the introduction of peptides or other non-permeant molecules, and the transfer of small electroporated peptides via gap junctions to inhibit the signal in the adjacent, non-electroporated cells is a powerful demonstration of signal inhibition.

This presentation demonstrates a method whereby electroporation of adherent, cultured cells is used for the study of intercellular, junctional communication, while the cells grow on a slide coated with conductive and transparent indium-tin oxide.

In this technique, cells are cultured on a glass slide that is partly coated with indium-tin oxide (ITO), a transparent, electrically conductive material. ...

Efficient and Cost Effective Electroporation Method to Study Primary Cilium-Dependent Signaling Pathways in the Granule Cell Precursor

The primary cilium is a critical signaling organelle found on nearly every cell that transduces Hedgehog (Hh) signaling stimuli from the cell surface. In the granule cell precursor (GCP), the primary cilium acts as a pivotal signaling center that orchestrates precursor cell proliferation by modulating the Hh signaling pathway. The investigation of primary cilium-dependent Hh signaling machinery is facilitated by in vitro genetic manipulation of the pathway components to visualize their dynamic localization to the primary cilium. However, transfection of transgenes in the primary cultures of GCPs using the currently known electroporation methods is generally costly and often results in low cell viability and undesirable transfection efficiency. This paper introduces an efficient, cost-effective, and simple electroporation protocol that demonstrates a high transfection efficiency of ~80-90% and optimal cell viability. This is a simple, reproducible, and efficient genetic modification method that is applicable to the study of the primary cilium-dependent Hedgehog signaling pathway in primary GCP cultures.

Here, we present a reproducible in vitro electroporation protocol for genetic manipulation of primary cerebellar granule cell precursors (GCPs) that is cost-effective, efficient, and viable. Moreover, this protocol also demonstrates a straightforward method for the molecular study of primary cilium-dependent Hedgehog signaling pathways in primary GCP cells.

The primary cilium is a critical signaling organelle found on nearly every cell that transduces Hedgehog (Hh) signaling stimuli from the cell surface. In ...

Electroporation-Mediated In Vitro Gene Delivery: An Electric Pulse Technique to Introduce Fluorescent Reporter Protein-Encoding Plasmids Into Cultured Cells

Source: Lo, J. C. W. et al., <a href="https://www.jove.com/v/63283">Efficient and Cost Effective Electroporation Method to Study Primary Cilium-Dependent Signaling Pathways in the Granule Cell Precursor.</a>&#160;J. Vis. Exp.&#160; (2021)
This video demonstrates the electroporation technique that introduces fluorescent protein-encoding plasmid DNA into mouse primary cerebellar granule cell precursors.

Source: Lo, J. C. W. et al., Efficient and Cost Effective Electroporation Method to Study Primary Cilium-Dependent Signaling Pathways in the Granule Cell ...

Monitoring Electroporation-Induced Changes in Action Potential Generation in Genetically Engineered Tet-On Spiking HEK cells

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