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EoE Sub Articles

1. Setting Up and Performing Transendothelial Electrical Resistance (TEER) Measurement
<ol>
	<li>Place the 24-well module of the transendothelial electrical resistance (TEER) instrument under the laminar flow hood. Remove the lids and place inserts with primary mouse brain microvascular endothelial cells (MBMECs) in the instrument using forceps.</li>
	<li>Pipette 810 &#181;l of fresh medium to the lower compartment of the module wells: add it carefully between the insert and the wall of the module well.</li>
	<li>Close the lids and place the instrument in the incubator. Connect the instrument to its computer; turn on the instrument controller and open the software.</li>
	<li>Select &#39;new measurement&#39; in the pop-up window. Check the &#39;show TEER&#39; and &#39;show Ccl&#39; boxes; then press &#39;start.&#39; After completing the first measurement, select &#39;check all wells&#39; in the &#39;results&#39; tab to see all TEER and cell layer capacitance (Ccl) values.</li>
	<li>Save the file: File&#62;Save as&#62;&#39; the name of your file&#39;. 
	NOTE: As TEER and Ccl are continuously measured in an automated fashion, monitor their values over a period of three to five days. Changing the medium is not necessary unless cell viability is suboptimal, as visualized by a lack of increase in TEER.</li>
	<li>Choose the time point to co-culture MBMECs with T cells when Ccl is stable and lower than 1 &#181;F/cm2 and TEER has reached its maximum level.</li>
	<li>Carefully inspect absolute TEER and Ccl values and exclude the wells in which MBMECs have not developed confluent enough monolayers by unchecking such wells.</li>
	<li>Group the rest of the wells: right-click on a well and select &#39;add well to new average well&#39;. Name it in the pop-up window; do the same for all individual wells to be grouped.</li>
	<li>Check all average wells to confirm that all of them have the same initial conditions before the co-culture. If some have significantly different absolute TEER values or TEER slopes, or the standard errors are too large, redo the grouping. 
	NOTE: The optimal grouping of wells provides minimal variation in TEER values within and between experimental groups.</li>
	<li>In the &#39;experiment&#39; tab, press &#39;pause&#39;, disconnect the instrument and take it out of the incubator. Remove the lids under the laminar flow hood.</li>
	<li>Prepare pre-activated and/or na&#239;ve T cells, with or without specific cytokines, antibodies, or other substances, as desired. For example, mix purified native/low endotoxin (NA/LE) rat anti-mouse interferon-gamma (IFN-&#947;) antibody (clone XGM1.2) with prepared T cells at 20 &#181;g/ml per well of the TEER instrument. 
	NOTE: If substances such as granzyme B inhibitor are used, they are added to T cell culture at the beginning of T cell stimulation: e.g., Granzyme B Inhibitor II (Calbiochem) is mixed with isolated T cells at a final concentration of 10 &#181;M in dimethyl sulfoxide (DMSO).</li>
	<li>Remove some of the media from the insert (the upper well compartment). Example: carefully pipette out 150 &#181;l (removing all the medium should be avoided as it could disturb the MBMEC monolayer).</li>
	<li>Add T cells to MBMECs by carefully pipetting 150 &#181;l of medium containing 2 x 105 cells/insert. 
	NOTE: When harvesting pre-activated T cells, count only blasting Trypan Blue-free cells.</li>
	<li>Close the lids and place the instrument back in the incubator. Reconnect the instrument and press &#39;resume measurement&#39;. After 24 hours, press &#39;stop&#39; in the &#39;experiment&#39; tab and save the file (File&#62;Save).</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>cellZscope</td>
			<td>nanoAnalytics GmbH</td>
			<td>www.nanoanalytics.com</td>
			<td>including: 24-well Cell Module, Controller, PC with cellZscope software v2.2.2</td>
		</tr>
		<tr>
			<td>Transwell membrane inserts - pore size 0.4 &#181;m</td>
			<td>Corning</td>
			<td>3470</td>
			<td>for TEER measurement as the main readout</td>
		</tr>
		<tr>
			<td>Transwell membrane inserts - pore size 3 &#181;m</td>
			<td>Corning</td>
			<td>3472</td>
			<td>for TEER measurement as the quality control prior to T-cell transmigration assay</td>
		</tr>
		<tr>
			<td>24-well cell culture plate</td>
			<td>Greiner</td>
			<td>650 180</td>
			<td>flat-bottom; for MBMEC culture</td>
		</tr>
		<tr>
			<td>96-well cell culture plate</td>
			<td>Costar</td>
			<td>3526</td>
			<td>round-bottom; for immune cell culture</td>
		</tr>
		<tr>
			<td>Neubauer counting chamber</td>
			<td>Marienfeld</td>
			<td>MF-0640010</td>
			<td>for cell counting</td>
		</tr>
		<tr>
			<td>Collagen type IV from human placenta</td>
			<td>Sigma</td>
			<td>C5533</td>
			<td>for MBMEC coating solution</td>
		</tr>
		<tr>
			<td>Fibronectin from bovine plasma</td>
			<td>Sigma</td>
			<td>F1141-5MG</td>
			<td>for MBMEC coating solution</td>
		</tr>
		<tr>
			<td>DMEM (+ GlutaMAX)</td>
			<td>Gibco</td>
			<td>31966-021</td>
			<td>for MBMEC isolation and MBMEC culture medium</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Sigma</td>
			<td>P4333</td>
			<td>for MBMEC isolation and MBMEC culture medium</td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Sigma</td>
			<td>H3393</td>
			<td>for MBMEC culture medium</td>
		</tr>
		<tr>
			<td>Human Basic Fibroblast Growth Factor (bFGF)</td>
			<td>PeproTech</td>
			<td>100-18B</td>
			<td>for MBMEC culture medium</td>
		</tr>
		<tr>
			<td>IMDM + 1% L-Glutamin</td>
			<td>Gibco</td>
			<td>21980-032</td>
			<td>for T cell culture medium</td>
		</tr>
		<tr>
			<td>Recombinant Murine IFN-&#947;</td>
			<td>PeproTech</td>
			<td>315-05</td>
			<td>for T-cell transmigration assays</td>
		</tr>
		<tr>
			<td>Recombinant Murine TNF-&#945;</td>
			<td>PeproTech</td>
			<td>315-01A</td>
			<td>for T-cell transmigration assays</td>
		</tr>
		<tr>
			<td>NA/LE purified anti-mouse IFN-&#947; antibody</td>
			<td>BD Biosciences</td>
			<td>554408</td>
			<td>clone XMG1.2; recommended final concentration: 20 &#181;g/ml</td>
		</tr>
		<tr>
			<td>Granzyme B Inhibitor II</td>
			<td>Calbiochem</td>
			<td>368055</td>
			<td>recommended final concentration: 10 &#181;M</td>
		</tr>
		<tr>
			<td>PE anti-mouse CD4 antibody</td>
			<td>Biolegend</td>
			<td>116005</td>
			<td>clone RM4-4; for analysis of T cell transmigration</td>
		</tr>
		<tr>
			<td>Trypan Blue solution, 0.4%</td>
			<td>Thermo Fisher Scientific</td>
			<td>15250061</td>
			<td>for cell counting</td>
		</tr>
	</tbody>
</table>

assessing transendothelial electrical resistance in an in vitro blood-brain barrier model

Source: Kuzmanov, I., et al. <a href="https://app.jove.com/v/54592">An In Vitro Model of the Blood-brain Barrier Using Impedance Spectroscopy: A Focus on T Cell-endothelial Cell Interaction.</a> J. Vis. Exp. (2016)
This video demonstrates a method for assessing transendothelial electrical resistance (TEER) in mouse brain microvascular endothelial cells in vitro. A high TEER value indicates intact endothelial cell junctions, while the addition of activated T cells lowers the TEER value, reflecting disruption of the junctional barrier.

Assessing Transendothelial Electrical Resistance in an In Vitro Blood-Brain Barrier Model

Source: Kuzmanov, I., et al. An In Vitro Model of the Blood-brain Barrier Using Impedance Spectroscopy: A Focus on T Cell-endothelial Cell Interaction. J. ...

Begin with a membrane insert containing mouse brain microvascular endothelial cells, resembling an in vitro blood-brain barrier.
Place this insert into a transendothelial electrical resistance or TEER instrument well containing an embedded electrode.
Add fresh media to the lower compartment.
Place the lid with electrodes over the TEER wells, then put the instrument in an incubator and connect it to a recording system.
The electrodes generate an electrical current across the cell layer for measurement of transendothelial resistance.
Intact junctional proteins between the cells impede the flow of current, resulting in a higher TEER value.
Disconnect the instrument and remove it.
Disassemble the lid, remove some medium from the upper compartment, and add pre-activated T cells. Reassemble the instrument for TEER measurement.
Activated T cells interact with the endothelial cells, releasing inflammatory factors and cytolytic enzymes, disrupting the junctional integrity.
This disruption increases electrical current flow, resulting in a lower TEER value.

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Neuropathology

Cerebrovascular Diseases

58 Aufrufe. Source: Kuzmanov, I., et al. An In Vitro Model of the Blood-brain Barrier Using Impedance Spectroscopy: A Focus on T Cell-endothelial Cell Interaction. J. Vis. Exp. (2016) This video demonstrates a method for assessing transendothelial electrical resistance (TEER) in mouse brain microvascular endothelial cells in vitro. A high TEER value indicates intact endothelial cell junctions, while the addition of activated T cells lowers the TEER value, reflecting disruption of the junctional barrier. 

Serie: Assessing Transendothelial Electrical Resistance in an In Vitro Blood-Brain Barrier Model - Konzept

Setting-up an In Vitro Model of Rat Blood-brain Barrier (BBB): A Focus on BBB Impermeability and Receptor-mediated Transport

The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and characterize a highly reproducible rat syngeneic in vitro model of the BBB using co-cultures of primary rat brain endothelial cells (RBEC) and astrocytes to study receptors involved in transcytosis across the endothelial cell monolayer. Astrocytes were isolated by mechanical dissection following trypsin digestion and were frozen for later co-culture. RBEC were isolated from 5-week-old rat cortices. The brains were cleaned of meninges and white matter, and mechanically dissociated following enzymatic digestion. Thereafter, the tissue homogenate was centrifuged in bovine serum albumin to separate vessel fragments from nervous tissue. The vessel fragments underwent a second enzymatic digestion to free endothelial cells from their extracellular matrix. The remaining contaminating cells such as pericytes were further eliminated by plating the microvessel fragments in puromycin-containing medium. They were then passaged onto filters for co-culture with astrocytes grown on the bottom of the wells. RBEC expressed high levels of tight junction (TJ) proteins such as occludin, claudin-5 and ZO-1 with a typical localization at the cell borders. The transendothelial electrical resistance (TEER) of brain endothelial monolayers, indicating the tightness of TJs reached 300 ohm&#183;cm2 on average. The endothelial permeability coefficients (Pe) for lucifer yellow (LY) was highly reproducible with an average of 0.26 &#177;&#160;0.11 x 10-3 cm/min. Brain endothelial cells organized in monolayers expressed the efflux transporter P-glycoprotein (P-gp), showed a polarized transport of rhodamine 123, a ligand for P-gp, and showed specific transport of transferrin-Cy3 and DiILDL across the endothelial cell monolayer. In conclusion, we provide a protocol for setting up an in vitro BBB model that is highly reproducible due to the quality assurance methods, and that is suitable for research on BBB transporters and receptors.

Setting-up an In Vitro Model of Rat Blood-brain Barrier BBB: A Focus on BBB Impermeability and Receptor-mediated Transport

The aim of the present study was to validate the reproducibility of an in vitro BBB model involving a rat syngeneic co-culture of endothelial cells and astrocytes. The endothelial cell monolayer presented high TEER and low LY permeability. Expression of specific TJ proteins, functional responses to inflammation and functionality of transporters and receptors were assessed.

Einrichten einer<em&gt; In-vitro-</em&gt; Modell der Ratte Blut-Hirn-Schranke (BBB): Ein Fokus auf BBB-Undurchlässigkeit und Rezeptor-vermittelten Transport

The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and ...

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JoVE Journal

Medizin

Measuring TEER in X-ray-Irradiated Epithelial Cells Co-cultured With PBMCs

To assess the transepithelial electrical resistance, or TEER, of the Caco-2 cells immediately after irradiation, transfer half of the Caco-2 cell culture inserts into a new plate with 3 milliliters of fresh complete medium in each well, and half of the insert cultures into a new plate seeded with 2 x 106 PBMC per 3 milliliters of complete medium per well. Then, place a TEER chopstick electrode into the cell culture insert every hour for the first six hours, and then every three hours until 48 hours after irradiation.

Assessing Transendothelial Electrical Resistance in an In Vitro Blood-Brain Barrier Model

Transkript

Assessing Transendothelial Electrical Resistance in an In Vitro Blood-Brain Barrier Model

Einrichten einer

Messung der TEER in röntgenbestrahlten Epithelzellen, die mit PBMCs kokultiviert wurden