Name: <meta charset="utf8"></head><body>מחבר זרקור: אופטימיזציה של אלקטרופורציה של מצע נקבובי באמצעות מיקרו וננו-ערוצים לניטור משופר ואפיון שלב ביניים
Uploaded: 2024-09-27T13:40:00
Description: Porous substrate electroporation (PSEP) is an emerging method of electroporation that provides high throughput and consistent delivery. Like many other ...

<meta charset="utf8"></head><body>שיטת TEEI לניתוח אלקטרופורציה וחיזוי מסירה בתאי A431

<ol>
	<li>Brooks, J., 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=High+throughput+and+highly+controllable+methods+for+in+vitro+intracellular+delivery.">High throughput and highly controllable methods for in vitro intracellular delivery.</a> Small. 16 (51), e2004917 (2020).</li><li>Stewart, M. P., Langer, R., Jensen, K. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+delivery+by+membrane+disruption:+Mechanisms,+strategies,+and+concepts.">Intracellular delivery by membrane disruption: Mechanisms, strategies, and concepts.</a> Chem Rev. 118 (16), 7409-7531 (2018).</li><li>Canatella, P. J., Karr, J. F., Petros, J. A., Prausnitz, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+study+of+electroporation-mediated+molecular+uptake+and+cell+viability.">Quantitative study of electroporation-mediated molecular uptake and cell viability.</a> Biophys J. 80 (2), 755-764 (2001).</li><li>Pliquett, U., Gift, E. A., Weaver, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+the+electric+field+and+anomalous+heating+caused+by+exponential+pulses+with+aluminum+electrodes+in+electroporation+experiments.">Determination of the electric field and anomalous heating caused by exponential pulses with aluminum electrodes in electroporation experiments.</a> Bioelectrochem Bioenerg. 39 (1), 39-53 (1996).</li><li>Pan, J., 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=Cell+membrane+damage+and+cargo+delivery+in+nano-electroporation.">Cell membrane damage and cargo delivery in nano-electroporation.</a> Nanoscale. 15 (8), 4080-4089 (2023).</li><li>Boukany, P. E., 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=Nanochannel+electroporation+delivers+precise+amounts+of+biomolecules+into+living+cells.">Nanochannel electroporation delivers precise amounts of biomolecules into living cells.</a> Nat Nanotechnol. 6 (11), 747-754 (2011).</li><li>Chang, L., 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=Micro-/nanoscale+electroporation.">Micro-/nanoscale electroporation.</a> Lab Chip. 16 (21), 4047-4062 (2016).</li><li>Patino, C. 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=Multiplexed+high-throughput+localized+electroporation+workflow+with+deep+learning-based+analysis+for+cell+engineering.">Multiplexed high-throughput localized electroporation workflow with deep learning-based analysis for cell engineering.</a> Sci Adv. 8 (29), eabn7637 (2022).</li><li>Sagvolden, G., Giaever, I., Pettersen, E. O., Feder, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+adhesion+force+microscopy.">Cell adhesion force microscopy.</a> Proc Natl Acad Sci U S A. 96 (2), 471-476 (1999).</li><li>Ishibashi, T., Takoh, K., Kaji, H., Abe, T., Nishizawa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+porous+membrane-based+culture+substrate+for+localized+in+situ+electroporation+of+adherent+mammalian+cells.">A porous membrane-based culture substrate for localized in situ electroporation of adherent mammalian cells.</a> Sensors Actuators B: Chem. 128 (1), 5-11 (2007).</li><li>Mukherjee, P., Nathamgari, S. S. P., Kessler, J. A., Espinosa, H. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combined+numerical+and+experimental+investigation+of+localized+electroporation-based+cell+transfection+and+sampling.">Combined numerical and experimental investigation of localized electroporation-based cell transfection and sampling.</a> ACS Nano. 12 (12), 12118-12128 (2018).</li><li>Brooks, J. R., 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=An+equivalent+circuit+model+for+localized+electroporation+on+porous+substrates.">An equivalent circuit model for localized electroporation on porous substrates.</a> Biosens Bioelectron. 199, 113862 (2022).</li><li>Brooks, J. R., 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=Transepithelial+electrical+impedance+increase+following+porous+substrate+electroporation+enables+label-free+delivery.">Transepithelial electrical impedance increase following porous substrate electroporation enables label-free delivery.</a> Small. 20 (25), 2310221 (2023).</li><li>Vindi&#353;, 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=Gene+electrotransfer+into+mammalian+cells+using+commercial+cell+culture+inserts+with+porous+substrate.">Gene electrotransfer into mammalian cells using commercial cell culture inserts with porous substrate.</a> Pharmaceutics. 14 (9), 1959 (2022).</li><li>Ye, 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=Single-cell+electroporation+with+real-time+impedance+assessment+using+a+constriction+microchannel.">Single-cell electroporation with real-time impedance assessment using a constriction microchannel.</a> Micromachines. 11 (9), 856 (2020).</li><li>Bednarek, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+methods+for+measuring+the+permeability+of+cell+monolayers.">In vitro methods for measuring the permeability of cell monolayers.</a> Methods Protoc. 5 (1), 17 (2022).</li><li>Harhaj, N. S., Antonetti, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+tight+junctions+and+loss+of+barrier+function+in+pathophysiology.">Regulation of tight junctions and loss of barrier function in pathophysiology.</a> Int J Biochem Cell Biol. 36 (7), 1206-1237 (2004).</li><li>Hunter, 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=.">.</a> Zeta potential in colloid science: Principles and applications. Vol. 2, (2013).</li><li>Wong, P. K., Wang, T. H., Deval, J. H., Ho, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrokinetics+in+microdevices+for+biotechnology+applications.">Electrokinetics in microdevices for biotechnology applications.</a> IEEE/ASME Trans Mechatron. 9 (2), 366-376 (2004).</li><li>Qian, K., Wang, Y., Lei, Y., Yang, Q., Yao, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+experimental+and+theoretical+study+on+cell+swelling+for+osmotic+imbalance+induced+by+electroporation.">An experimental and theoretical study on cell swelling for osmotic imbalance induced by electroporation.</a> Bioelectrochemistry. 157, 108637 (2024).</li><li>Fox, M. B., 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+of+cells+in+microfluidic+devices:+A+review.">Electroporation of cells in microfluidic devices: A review.</a> Anal Bioanal Chem. 385 (3), 474-485 (2006).</li></ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>15 mL Conical Centrifuge Tube</td>
			<td>Thermo Scientific</td>
			<td>339651</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Chip Disposable Hemocytometer</td>
			<td>Bulldog Bio</td>
			<td>DHC-N01</td>
			<td></td>
		</tr>
		<tr>
			<td>75 cm2 Tissue Culture Flask</td>
			<td>fisherbrand</td>
			<td>FB012937</td>
			<td></td>
		</tr>
		<tr>
			<td>A431 Cells</td>
			<td>ATCC</td>
			<td>CRL-1555</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcein AM</td>
			<td>Invitrogen</td>
			<td>C3099</td>
			<td></td>
		</tr>
		<tr>
			<td>Class II Type A2 Biosafety Cabinet</td>
			<td>Labgard</td>
			<td>NU-543-600</td>
			<td></td>
		</tr>
		<tr>
			<td>Custom Components</td>
			<td>YangLab</td>
			<td></td>
			<td>https://github.com/YangLabUNL/PSEP-TEEI</td>
		</tr>
		<tr>
			<td>Disposable Centrifuge Tube (50 mL)</td>
			<td>fisherbrand</td>
			<td>05-539-6</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco</td>
			<td>11965092</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Gibco</td>
			<td>A5670401</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluid Aspiration System</td>
			<td>vacuubrand</td>
			<td>20727403</td>
			<td></td>
		</tr>
		<tr>
			<td>HERACELL 240i</td>
			<td>Thermo Scientific</td>
			<td>51026331</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst 33342</td>
			<td>Thermo Scientific</td>
			<td>62249</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Plasma Fibronectin</td>
			<td>Sigma-Aldrich</td>
			<td>FIBRP-RO</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted Fluorescent Microscope</td>
			<td>Zeiss</td>
			<td>491916-0001-000</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted Microscope</td>
			<td>Labomed</td>
			<td>TCM 400</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>cytiva</td>
			<td>SH30256.02</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR Tube 200 &#181;L</td>
			<td>Sarstedt</td>
			<td>72.737</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin / Streptomycin</td>
			<td>Gibco</td>
			<td>15140148</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette (0.2-2 &#181;L)</td>
			<td>fisherbrand Elite</td>
			<td>FBE00002</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette (100-1000 &#181;L)</td>
			<td>fisherbrand Elite</td>
			<td>FBE01000</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette (20-200 &#181;L)</td>
			<td>fisherbrand Elite</td>
			<td>FBE00200</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette (2-20 &#181;L)</td>
			<td>fisherbrand Elite</td>
			<td>FBE00020</td>
			<td></td>
		</tr>
		<tr>
			<td>Propidium Iodide</td>
			<td>Invitrogen</td>
			<td>P1304MP</td>
			<td></td>
		</tr>
		<tr>
			<td>Reaction Tube 1.5 mL</td>
			<td>Sarstedt</td>
			<td>72.690.300</td>
			<td></td>
		</tr>
		<tr>
			<td>Sorvall ST 16R Centrifuge</td>
			<td>Thermo Scientific</td>
			<td>75004240</td>
			<td></td>
		</tr>
		<tr>
			<td>Thincert (24-well)</td>
			<td>Greiner Bio-One</td>
			<td>662 641</td>
			<td>0.4 &#181;m pore diameter, 2x106 cm-2 pore density, transparent PET</td>
		</tr>
		<tr>
			<td>Tissue Culture Plate (24-well)</td>
			<td>fisherbrand</td>
			<td>FB012929</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue Solution</td>
			<td>Sigma-Aldrich</td>
			<td>T8154-20mL</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Gibco</td>
			<td>15090046</td>
			<td></td>
		</tr>
	</tbody>
</table>

porous substrate-based electroporation with transepithelial electrical impedance monitoring

Electroporation is a technique in which cells are exposed to an electric field, creating temporary pores in the cell membrane through which cargos, including proteins, RNA, and DNA, can pass1,2. The most widely used version is bulk electroporation (BEP). BEP is performed by filling a cuvette with an electrolyte containing millions of cells, exposing the electrolyte to high voltage, and allowing cargo to enter the cells through diffusion or endocytosis1. There are many advantages to BEP, including high throughput and numerous commercially available systems. However, there are limitations to the BEP delivery. Inconsistent cell positioning relative to the electrodes and electric field shielding from adjacent cells causes significant variability in electric field exposure during BEP3,4. The high voltage required for BEP also has a significant negative impact on cell viability5. Since its inception in 20116, there has been growing interest in an electroporation method called porous substrate electroporation (PSEP), though it is sometimes referred to by other names, including localized electroporation and nano- or micro-electroporation1,7,8. In contrast to the cell suspension of BEP, PSEP is conducted on cells that are adherent to a porous substrate. Not only is an adherent state preferred for the majority of human cell lines9, but the pores in the substrate also focus on the electric current, localizing the transmembrane electrical potential (TMP) to specific regions of the cell membrane10,11. This localization allows for a significant reduction in applied voltage, decreasing damage and increasing cell viability. This combination of effects helps control cell membrane pore development, resulting in a more consistent and efficient delivery1,5,12.
A recent study introduced a PSEP device with a six-well, gold-plated electrode array for holding commercially available porous membrane inserts13 (Figure 1A,B), a practice that was first introduced by Vindis et al.14. The device can apply pulses and measure the electrical impedance across the cell monolayer, known as the transepithelial electrical impedance (TEEI), in real-time13. The user interface of the device allows complete control over the electroporation waveform and polarity. Importantly, real-time impedance measurements can be used to predict delivery outcomes without the need for expensive reagents or fluorescent markers, a concept known as label-free delivery15.
The PSEP platform consists of two major custom electrical components: the main body of the device, which houses the pulse generator and TEEI measurement equipment, and the electrode array, where the porous substrates are inserted, and the electroporation occurs. Diagrams for all custom electronics and 3D-printed components can be found at GitHub:&#160;https://github.com/YangLabUNL/PSEP-TEEI. In addition to the custom electronics, a computer is also required for the platform to function properly. The custom software requires MATLAB (version 2021a or later) to run, and Microsoft Excel to store and access data for analysis. The program controls the custom electronics and provides the graphical user interface (GUI) for adjusting settings. These programs were also made available at GitHub: https://github.com/YangLabUNL/PSEP-TEEI.
Preliminary data suggests this process is possible for different types of adherent cells (Figure 1C), but this article will only discuss the preparation of A431 cells using parameters that were found to be optimal for this cell line by Brooks et al.13. Additionally, because the propidium iodide (PI) cargo is cytotoxic, two experiments are performed, the first with a high concentration PI transfection media to quantify delivery efficiency, and the second with only cell culture media to measure TEEI over longer timescales. These experiments use identical electroporation waveforms, allowing the results to be correlated (Figure 1D).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66971/66971fig01.jpg" /> 
Figure 1: Electrode array assembly diagram and foundational data. (A) CAD model of an insert inside a well of the electrode array. (B) CAD model of the electrode array. (C) Impedance increase due to PSEP for select cell lines, n = 3 per cell line. Error bar: standard error of the mean. (D) Delivery efficiency vs. TEEI increase correlation data. Delivery efficiency was calculated by dividing the number of cells labeled in both PI and calcein images from delivery experiments by the total number of cells identified with Hoechst. Cell count was determined using a custom CellProfiler pipeline, n = 6 per voltage. Error bar: (x- and y-axis) standard error of the mean. This figure is reproduced from Brooks et al.13 with permission. <a href="https://www.jove.com/files/ftp_upload/66971/66971fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<meta charset="utf8"></head><body>מחבר זרקור: אופטימיזציה של אלקטרופורציה של מצע נקבובי באמצעות מיקרו וננו-ערוצים לניטור משופר ואפיון שלב ביניים

אלקטרופורציה מבוססת מצע נקבובי עם ניטור עכבה חשמלית טרנסאפיתל

Our research is focused on improving porous substrate electroporation. Research has shown major benefits from using micro and nano channels to localize electroporation. But there are still questions to be answered, especially regarding monitoring and characterizing effects during the intermediate stages of the process.Some of the most recent developments include our use of trans-epithelial electrical impedance monitoring, as well as using nano channel based electroporation to create exosomes or measure tension in cell membranes. PSEP experiments still rely heavily on before and after fluorescent imaging to determine delivery and cell viability outcomes. Additionally, many PSEP based studies under report pulse parameters and very little research has been done to verify optimal parameters for common cell types.The primary research gap we address is the lack of reporting on the effects of PSEP during the cell recovery stage. However, our analysis of TEEI measurements correlated TEEI change with viability and delivery efficiency, which allowed us to identify optimal PSEP waveform parameters as well. Our primary focus moving forward will be to identify the mechanisms causing the TEEI increase.Ideally, those findings will help us fine tune the PSEP process for an even more efficient delivery.

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

porous-substrate-based-electroporation-with-transepithelial

Research

JoVE Journal

Bioengineering

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.

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.

Fibronectin Coating and A431 Cell Seeding for Porous Substrate-Based Electroporation

To begin fibronectin coating, select 12 inserts and 2 24-well plates. Place the inserts into one well plate, creating two rows of six. Prepare one microgram per milliliter fibronectin solution by mixing 52 microliters of fibronectin stock solution and 1.2 milliliters of PBS in a 1.5 milliliter tube.Distribute 100 microliters of the fibronectin solution into each insert. Incubate the inserts in the well plate at 37 degrees Celsius for three hours. After incubation, remove the excess fibronectin solution from each insert.Wash the inserts twice by adding 100 microliters of sterile distilled water to each insert. Remove the water in the same order as it was added to ensure consistent wash time between inserts. Now wash the inserts with 100 microliters of cell culture media, and then remove the media from the inserts.Next, seed 200 microliters of A431 cell suspension into the inserts. For negative control, pipette 200 microliters of cell culture media into the inserts. Using a permanent marker, draw a line dividing the second well plate into two columns that are three wells wide.Separate each column into rows. Label each region in the grid with relevant parameters. Add one milliliter of cell culture media to each well.Transfer the inserts from the preparation plate to their appropriate location in the labeled experiment plate and incubate at 37 degrees Celsius for 12 hours.

ציפוי פיברונקטין וזריעת תאי A431 לאלקטרופורציה על בסיס מצע נקבובי

Transepithelial Electrical Impedance (TEEI) Monitoring in A431 Cells Using Electrode Array

To begin, pipette 1.5 milliliters of the 0.1 milligram per milliliter propidium iodide solution into each well in the electrode array. Place an insert into each well in the electrode array, fitting the feet of the insert into the alignment grooves so the insert is flush with the upper surface of the well. Screw the top electrode printed circuit board to the top of the electrode array wells and connect the electrode array to the porous substrate electroporation, or PSEP device.Place the electrode array in the 37 degrees Celsius incubator for temperature equilibration. Click the dropdown next to membrane in the top left corner of the GUI and click on 400 nanometer GBO. On the right side of the GUI, type one into the post pulse time duration minute edit field.Now, click on the run button and enter appropriate names for wells one to three and four to six when prompted. Then, click on okay to start the experiment. After one hour, remove the electrode array from the incubator and transfer the inserts back into the original locations in the experiment plate.Mix two microliters of Hoechst 33342 and five microliters of calcein AM with 123 microliters of cell culture media in a 200 microliter tube. Gently pipette 10 microliters of the stain solution into each post pulse insert and place the inserts back into the incubator for five minutes. Transfer the well plate to the plate holder of a fluorescent microscope with a 5x magnification objective.Image using brightfield and the fluorescence of each stain. Pipette 1.5 milliliters of the cell culture media into each well in the electrode array. Place cell sample inserts into wells one to three and control inserts into wells four to six, fitting the feet of the insert into the alignment grooves.Screw the top electrode printed circuit board to the top of the electrode array wells and connect the electrode array to the PSEP device. Place the electrode array in the 37 degrees Celsius incubator for temperature equilibration. Click on the dropdown next to membrane in the top left corner of the GUI and click on 400 nanometer GBO.Now, click on the run button and enter appropriate names for wells one to three and four to six when prompted. Then, click on okay to start the experiment. After one hour, remove the electrode array from the incubator and transfer the inserts back into the original locations in the experiment well plate.After preparing Hoechst 33342, calcein AM, and propidium iodide in cell culture media, mix and gently pipette 10 microliters of the stain solution into each post pulse insert, and place the inserts back into the incubator for five minutes. On a fluorescent microscope, image the cell sample inserts using brightfield and the fluorescence of each stain. The optimized healthy curve showed no dip below the baseline and the largest increase in TEEI.Post PSEP imaging of the cell monolayer revealed negligible cell death and a healthy fully confluent cell monolayer. The optimized unhealthy data resulted in a diminished TEEI response. Post PSEP imaging revealed near zero cell death and reduced delivery efficiency due to fewer cells in the monolayer.Application of unoptimized waveforms resulted in significant decreases in TEEI response, indicating substantial cell death and diminished delivery efficiency.