We are grateful to Annika Engbers and Frank Kurth for their excellent technical support and Dr. Markus Sch&#228;fer (nanoAnalytics GmbH) for helpful discussions regarding TEER measurements. This work was supported by the Deutsche Forschungsgemeinschaft (DFG), SFB1009 project A03 to HW and LK, CRC TR128, projects A08; Z1 and B01 to LK and HW, and the Interdisciplinary Center for Clinical Research (Medical Faculty of M&#252;nster) grant number Kl2/2015/14 to LK.

For all experiments, mice were bred and maintained under specific pathogen-free conditions in the central animal facility at the University of M&#252;nster, according to German guidelines for animal care. All experiments were performed according to the guidelines of the animal experimental ethics committee and approved by the local authorities of North Rhine-Westphalia, Germany (LANUV, AZ 84-02.05.20.12.217).
1. MBMEC Isolation and Culture
NOTE: Isolate MBMECs as previously described in detail14 with the following modifications:
<ol>
	<li>Sacrifice 20 adult C57BL/6 mice (6-8 weeks old) by CO2 inhalation and confirm their death by ascertaining cardiac and respiratory arrest.</li>
	<li>Rinse the mouse with 70% ethanol and decapitate it using scissors; remove the scalp using forceps and scissors. Make incisions on the left and right side of the skull, starting from the foramen magnum. Lift the skull from its caudal side and take out the brain with forceps.</li>
	<li>Under a laminar flow hood, use sterile forceps to place the brain in a Petri dish and remove the brain stem, cerebellum, and thalamus, keeping only the cortex. 
	NOTE: Use of a microscope is not necessary for any of these steps.</li>
	<li>Place the cortex onto a piece of sterile Western blotting paper and roll it gently with forceps until meninges are no longer visible.</li>
	<li>After mechanical and enzymatic digestion (following the protocol14), collect endothelial cells from the density gradient by using a long, sterile needle and a 5 ml plunger. Endothelial cells appear as a murky layer above the red ring with erythrocytes. Transfer 20-25 ml of this layer into a new 50 ml centrifugation tube; top up the tube with DMEM.</li>
	<li>After two rounds of washing14, resuspend the cells in 6 ml of MBMEC medium containing Puromycin (4 &#181;g/ml) and seed them onto six coated wells of a 24-well plate; leave them in a tissue culture incubator at 37 &#176;C and 5% CO2 (referred to as &#39;incubator&#39; from here on) for three days. 
	NOTE: For details of MBMEC coating solution, see Materials Table.</li>
	<li>Change the medium by adding 1 ml to each well of fresh MBMEC medium without Puromycin; put the plate back at 37 &#176;C and 5% CO2 for two more days.</li>
</ol>
2. Harvesting MBMECs
<ol>
	<li>On day five after MBMEC isolation, pre-coat permeable inserts with MBMEC coating solution: add 80 &#181;l of solution per insert and leave them at 37 &#176;C and 5% CO2 for 3 hr.</li>
	<li>Carefully pipette out the coating solution and let the inserts air-dry for 30 min. Add 2 ml of room temperature (RT) PBS to each well of the plate, using MBMECs to wash the medium; repeat this step. Add 0.05% Trypsin-EDTA (300 &#181;l per well) and leave the plate in the incubator for 5-10 min.</li>
	<li>Add 2 ml of MBMEC medium to each well to stop trypsinization. Transfer the collected cells to a 15 ml centrifugation tube and centrifuge them at 700 x g for 8 min at 4 &#176;C. Discard the supernatant and resuspend the cells in 1 ml of MBMEC medium without Puromycin.</li>
	<li>Count the cells; mix 10 &#181;l of cell suspension with 90 &#181;l of 0.04% Trypan Blue (10x dilution of cells). Pipette 10 &#181;l of this mix between the glass cover and the cell counting chamber (hemocytometer). Count Trypan Blue-free cells in all four quadrants of the chamber (N) and determine the average number of counted cells (N&#39;) as follows: N&#39; = N/4.</li>
	<li>Calculate the cell concentration (in 106 cells/ml) using the following formula: C = N&#39; x 104 x 10, where 104 is given by the dimensions of the hemocytometer and 10 is the dilution factor from step 2.4.</li>
	<li>Seed 2 x 104 cells in a volume of 260 &#181;l per insert. Add 810 &#181;l of MBMEC medium to the lower compartment of each well. Place the plate with inserts in the incubator until ready to proceed with section 4. 
	NOTE: The volumes for the upper and lower compartment are insert-specific and do not work for all 24-well formats.</li>
</ol>
3. CD4+ T Cell Isolation and Stimulation
<ol>
	<li>CD4+ T Cell Isolation
	<ol>
		<li>Sacrifice one adult mouse (6-8 weeks old) by CO2 inhalation and confirm its death by ascertaining cardiac and respiratory arrest.</li>
		<li>Place the mouse on its back on a clean dissection board and rinse it with 70% ethanol; use sterile scissors and forceps to open the peritoneum and remove the spleen and lymph nodes (inguinal, axillary, brachial and cervical); finally, transfer the tissue into a 15 ml centrifugation tube containing 5 ml of PBS on ice.</li>
		<li>Under the laminar flow hood, transfer the tissue by decanting PBS with the tissue into a 50 ml centrifugation tube with a 70 &#181;m cell strainer on top; homogenize the tissue by pressing it with a 1 ml plunger through the strainer.</li>
		<li>Add 30 ml of FACS buffer and centrifuge it at 500 x g for 5 min at 4 &#176;C. Discard supernatant and resuspend the cells in 10 ml of FACS buffer. Filter the suspension through a 40 &#181;m cell strainer, add another 20 ml of FACS buffer to the tube.</li>
		<li>Centrifuge it at 500 x g for 5 min at 4 &#176;C. Discard the supernatant and resuspend the cells in 500 &#181;l of FACS buffer.</li>
		<li>Add 20 &#181;l of mouse CD4 magnetic microbeads, mix well and incubate for 15 min at 4 &#176;C. Add 25 ml of FACS buffer and centrifuge it at 500 x g for 5 min at 4 &#176;C.</li>
		<li>In the meantime, place an LS separation column into the magnet and rinse it with 3 ml of FACS buffer. Discard the supernatant and resuspend the cells in 3 ml of FACS buffer.</li>
		<li>Add cells to the column. Wash the column with 3 ml of FACS buffer three times. Remove the column from the magnet and place it onto a new 15 ml centrifugation tube. Add 5 ml of FACS buffer, flush out the labelled cells with the column plunger and centrifuge it at 500 x g for 5 min 4 &#176;C.</li>
		<li>Discard the supernatant and resuspend the cells in 3 ml of T cell medium. Count the cells as described in 2.4 and seed 1 x 105 cells in 100 &#181;l of medium per well.</li>
	</ol>
	</li>
	<li>CD4+ T Cell Stimulation
	<ol>
		<li>Polyclonal CD4+ T cell stimulation
		<ol>
			<li>Pre-coat a round-bottom 96-well plate with purified anti-mouse CD3 antibody (clone 145-2C11) in PBS, at a desired final concentration. For example, to pre-coat the full plate with &#945;-CD3 at 1 &#181;g/ ml, mix 10 &#181;l of the antibody (stock concentration = 0.5 mg/ml) with 5 ml of PBS, vortex and add 50 &#181;l of the mix to each well with a multichannel pipette.</li>
			<li>Leave the plate in the incubator for 3 hr. After isolating the T cells, wash the pre-coated plate twice with PBS. Add purified anti-CD28 antibody (clone 37.51) to isolated T cells at a desired final concentration (e.g., at 1 &#181;g/ml); mix well. Seed the T cells and leave them in the incubator for two to three days.</li>
		</ol>
		</li>
		<li>Antigen-specific CD4+ T cell stimulation with dendritic cells (DCs) 
		NOTE: If DCs are used as antigen-presenting cells (APCs), follow the Protocol for T cell isolation, with these exceptions:
		<ol>
			<li>Before homogenizing the spleen, inject it with 1 ml of Collagenase type IA in PBS at 0.5 mg/ml and transfer it to a 15 ml centrifugation tube.</li>
			<li>Incubate in the water bath at 37 &#176;C for 15 min. After washing with PBS, resuspend the pellet in FACS buffer and add 20 &#181;l of mouse CD11c magnetic microbeads, instead of CD4 microbeads.</li>
			<li>Use an MS separation column and the appropriate volumes: rinse the column with 1 ml of FACS buffer; resuspend cells in 1 ml of FACS buffer and wash the column with 1 ml of FACS buffer three times.</li>
			<li>Add antigen of choice to DCs (e.g., myelin oligodendrocyte glycoprotein (MOG) a.a. 35-55 at 20 &#181;g/ml), mix well and seed 5 x 104 cells in 100 &#181;l of T cell culture medium per 96-round-bottom-well.</li>
			<li>Add 1 x 105 T cells in 100 &#181;l of T cell culture medium per 96-round-bottom-well at the end, as described in the Protocol for T cell isolation.</li>
		</ol>
		</li>
		<li>Antigen-specific CD4+ T cell stimulation with B cells 
		NOTE: If B cells are used as APCs, follow the Protocol for T cell isolation, with these exceptions:
		<ol>
			<li>Use spleens from transgenic mice whose B cells are antigen-specific (e.g., IgHMOG (Th) mice, whose B cells specifically recognize MOG35-55).</li>
			<li>Use antigen-specific T cells (e.g., from TCRMOG (2D2) mice). Add 20 &#181;l of mouse CD19 magnetic microbeads instead of CD4 microbeads.</li>
			<li>Add the antigen of choice to the B cells (e.g., MOG35-55 at 20 &#181;g/ml), mix well and seed 5 x 104 cells in 100 &#181;l of B cell culture medium per 96-round-bottom-well.</li>
			<li>Add 1 x 105 T cells in 100 &#181;l of T cell culture medium per 96-round-bottom-well, as described in the Protocol for T cell isolation.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
4. Setting Up and Performing TEER Measurement
<ol>
	<li>Place the 24-well module of the TEER instrument under the laminar flow hood. Remove the lids and place inserts with 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 &#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 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, both 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 NA/LE rat anti-mouse 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 DMSO. See Materials and Equipment for more details.</li>
	<li>Remove some of the medium from the insert (the upper well compartment): e.g., 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 to the incubator. Reconnect the instrument and press &#39;resume measurement&#39;. After 24 hr, press &#39;stop&#39; in the &#39;experiment&#39; tab and save the file (File&#62;Save).</li>
</ol>
5. Data Export and Statistical Analysis
<ol>
	<li>Export the results by choosing File&#62;Export.</li>
	<li>In the &#34;settings&#34; tab of the pop-up window, select &#34;.dot&#34; in the &#34;decimal separator&#34; option and &#34;tabulator&#34; in the &#34;field delimiter&#34; option.</li>
	<li>In the &#34;export results&#34; tab, name the file, check all the wells to be exported, and check the &#34;TEER&#34; and &#34;Ccl&#34; boxes in the &#34;choose data&#34; option.</li>
	<li>Press &#34;export data,&#34; open the exported file, and copy the data to a spreadsheet.</li>
	<li>Normalize the exported data (sorted by each replicate, with TEER and Ccl values given for each run (measurement)): Set TEER and Ccl values of the last time point before the co-culture to 100% and change accordingly the values for all other runs, relative to the &#39;100%&#39; run.</li>
	<li>Copy normalized data to a software of choice to generate a graph, using individual wells and displaying the standard error of the mean for each treatment group.</li>
	<li>Perform Two-way ANOVA statistical test with a Bonferroni correction for multiple comparisons, using the statistical software of choice.</li>
</ol>

I. Kuzmanov, A. M. Herrmann, S.G. Meuth, H. Wiendl and L. Klotz have nothing to disclose. H.-J. Galla is a scientific advisor of nanoAnalytics GmbH, the manufacturer of the automated cell monitor.

Several steps of the described protocol are essential for a successful experiment. During the initial MBMEC isolation and culture, it is crucial that work is performed under sterile conditions as much as possible, to prevent the contamination of the cell culture with fungal spores or bacteria. In order to obtain a pure culture of ECs, it is recommended to use a medium containing Puromycin for the first three days, which enables survival of ECs, but not other cells types (especially pericytes)41,42. Another cri...

The blood-brain barrier separates the systemic circulation from the central nervous system (CNS)1-3. It provides a physical barrier that inhibits the free movement of cells and the diffusion of water-soluble molecules and protects the brain from pathogens and potentially harmful substances. In addition to its barrier function, the BBB enables the delivery of oxygen and nutrients to the brain parenchyma, which ensures proper functioning of the neuronal tissue. Functional properties of the BBB are highly regulated by its cellular and acellular components, with highly specialized ECs being its main structural element. ECs of the BBB are characterized by the presence of tight junction (TJ) complexes, the lack of fenestrations, extremely low pinocytic activity, and permanently active transport mechanisms. Other components of the BBB the EC basement membrane, pericytes embedding the endothelium, astrocytic end feet and = associated parenchymal basement membrane also contribute to the development, maintenance and function of the BBB2,4-6 and, together with neurons and microglia, form the neurovascular unit (NVU), which enables proper functioning of the CNS7-9.
In a variety of neurological diseases, such as neurodegenerative, inflammatory or infectious diseases, the function of the BBB is compromised2,5,10. Dysregulation of TJ complexes and molecular transport mechanisms leads to increased BBB permeability, leukocyte extravasation, inflammation and neuronal damage. In order to study BBB properties under such pathophysiological conditions, various in vitro BBB models have been established9,11,12. Together they have provided valuable insights into the changes of barrier integrity, permeability as well as transport mechanisms. These models employ endothelial cells of human, mouse, rat, porcine or bovine origin13-18; primary endothelial cells or cell lines are cultured either as a monoculture or together with pericytes and/or astrocytes in order to mimic more closely the BBB in vivo19-25. In recent years, measurement of transendothelial electrical resistance (TEER) has become a widely accepted tool to assess endothelial barrier properties26,27.
TEER reflects the impedance to the ion flux across the cell monolayer and its decrease provides a sensitive measure of compromised endothelial barrier integrity and hence increased permeability. Various TEER measurement systems have been developed, including Epithelial Voltohmmeter (EVOM), Electric Cell-substrate Impedance Sensing (ECIS), and real-time cell analysis15,28-30. TEER reflects the resistance to the ion flux between adjacent ECs (paracellular route) and is directly proportional to the barrier integrity. In impedance spectroscopy27,31, complex total impedance (Z) is measured, which provides additional information about the barrier integrity by measuring Ccl. Ccl relates to the capacitive current through the cell membrane (transcellular route): the cell layer acts like a capacitor in the equivalent electric circuit, separating the charges on both sides of the membrane and is inversely proportional to the barrier integrity. When grown on permeable inserts, ECs adhere, proliferate and spread over the microporous membrane. This resists the background capacitive current of the insert (which itself acts like a capacitor)&#160;and leads to a decrease in the capacitance until it reaches its minimal level. This is followed by the establishment of TJ complexes that seal off the space between adjacent ECs. This restricts the ion flux through the paracellular route, and TEER increases until it reaches its plateau. Under inflammatory conditions, however, the endothelial barrier is compromised: TEER decreases as TJ complexes get disrupted and Ccl increases as the capacitive component of the insert rises again.
Our TEER measurement uses the automated cell monitoring32 system: it follows the principle of impedance spectroscopy and extends its previous applications. Here, we describe an in vitro BBB model that enables the study of the barrier properties, including the interaction of brain endothelium with immune cells; in particular activated T cells. Such pathophysiological conditions are observed in autoimmune diseases of the CNS, such as multiple sclerosis and its animal model experimental autoimmune encephalomyelitis33-37. Here, a crucial step is the transmigration of encephalitogenic, myelin-specific T cells across the BBB. This is followed by their reactivation in the perivascular space and entry into the brain parenchyma, where they recruit other immune cells and mediate inflammation and subsequent demyelination1,35,38. However, molecular mechanisms of the interaction between such T cells and endothelial cells, the main constituents of the BBB, are not well understood. Our protocol aims to fill this gap and give new insights into the consequences on endothelial cells (i.e., barrier integrity and permeability) upon their direct contact and complex interplay with activated T cells.
The protocol described here makes use of primary mouse brain microvascular endothelial cells, grown as a monolayer on permeable inserts with microporous membranes. Endothelial cells are co-cultured with CD4+ T cells, which can be pre-activated either polyclonally or in an antigen-specific fashion. Co-culture of MBMECs with pre-activated, but not na&#239;ve T cells induces a decrease in TEER and an increase in Ccl, which provides a quantitative measure of the MBMEC dysfunction and barrier disruption. The technique is non-invasive: it uses built-in instead of chopstick electrodes, which prevent major disturbance of the EC monolayer; it can be used to monitor barrier function without the use of cell markers. It makes continuous measurements in an automated fashion and enables an independent assessment of the two barrier parameters (TEER and Ccl) simultaneously over time. The method is also sensitive enough to distinguish between different levels of T cell activation and effects of such T cells on ECs.
It can be used in a wide range of functional assays: different cytokines and/or chemokines implicated in inflammatory processes can be added to the co-culture of MBMECs and T cells; blocking antibodies against cell adhesion molecules on either the EC or T-cell side can be used; and inhibitors of T cell activation markers or of their cytolytic properties can be added during the T-cell priming or their co-culture with ECs. The assay is also useful for T-cell transmigration assays, as it can serve as a quality control of the MBMEC monolayer integrity prior to the addition of T cells. All this makes this method a versatile and reliable tool to study the BBB in vitro, with a focus on the effect of activated T cells on EC monolayer integrity. This is of particular importance for understanding the mechanisms of the BBB disruption in the pathogenesis of autoimmune diseases, such as MS and its animal model EAE, where self-reactive, encephalitogenic T cells cross the BBB and cause inflammation and neuronal damage.

<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&#160;</td>
		</tr>
		<tr>
			<td>Ultracentrifuge</td>
			<td>Thermo Scientific</td>
			<td>www.thermoscientific.com</td>
			<td>SORVALL RC 6+; rotor F21S-8x50y; for MBMEC isolation</td>
		</tr>
		<tr>
			<td>flow cytometer</td>
			<td>Beckman Coulter</td>
			<td>www.beckmancoulter.com</td>
			<td>for analysis of T cell transmigration</td>
		</tr>
		<tr>
			<td>FlowJo7.6.5 software</td>
			<td>Tree Star</td>
			<td>www.flowjo.com</td>
			<td>for analysis of T cell transmigration</td>
		</tr>
		<tr>
			<td>Oak Ridge centrifuge tubes, PC</td>
			<td>Thermo Fisher Scientific</td>
			<td>3118-0050</td>
			<td>50 ml; for MBMEC isolation</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>QuadroMACS Separator</td>
			<td>Miltenyi Biotec</td>
			<td>130-090-976</td>
			<td>for T cell and B cell isolation; supports MACS LS columns</td>
		</tr>
		<tr>
			<td>OctoMACS Separator</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-109</td>
			<td>for dendritic cell isolation; supports MACS MS columns</td>
		</tr>
		<tr>
			<td>Neubauer counting chamber</td>
			<td>Marienfeld</td>
			<td>MF-0640010</td>
			<td>for cell counting</td>
		</tr>
		<tr>
			<td>Cell strainer, 70 &#181;m</td>
			<td>Corning</td>
			<td>352350</td>
			<td>for immune cell isolation</td>
		</tr>
		<tr>
			<td>Cell strainer, 40 &#181;m</td>
			<td>Corning</td>
			<td>352340</td>
			<td>for immune cell isolation</td>
		</tr>
		<tr>
			<td>MACS MultiStand</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-303</td>
			<td>for immune cell isolation</td>
		</tr>
		<tr>
			<td>MACS LS separation columns</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-401</td>
			<td>for T cell and B cell isolation</td>
		</tr>
		<tr>
			<td>MACS MS separation columns</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-201</td>
			<td>for dendritic cell isolation</td>
		</tr>
		<tr>
			<td>Mouse CD4 MicroBeads</td>
			<td>Miltenyi Biotec</td>
			<td>130-049-201</td>
			<td>for CD4+ T cell isolation</td>
		</tr>
		<tr>
			<td>Mouse CD19 MicroBeads</td>
			<td>Miltenyi Biotec</td>
			<td>130-052-201</td>
			<td>for B cell isolation</td>
		</tr>
		<tr>
			<td>Mouse CD11c MicroBeads</td>
			<td>Miltenyi Biotec</td>
			<td>130-052-001</td>
			<td>for dendritic cell isolation</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>Collagenase 2 (CSL2)</td>
			<td>Worthington</td>
			<td>LS004176</td>
			<td>for MBMEC isolation</td>
		</tr>
		<tr>
			<td>Collagenase/Dispase (C/D)</td>
			<td>Roche</td>
			<td>11097113001</td>
			<td>for MBMEC isolation</td>
		</tr>
		<tr>
			<td>DNase I</td>
			<td>Sigma</td>
			<td>DN25</td>
			<td>for MBMEC isolation</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Sigma</td>
			<td>F7524</td>
			<td>for MBMEC isolation</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Amresco</td>
			<td>0332-100G</td>
			<td>for MBMEC isolation</td>
		</tr>
		<tr>
			<td>Percoll</td>
			<td>Sigma</td>
			<td>P1644-1L</td>
			<td>for MBMEC isolation</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>Phosphate-Buffered Saline (PBS)</td>
			<td>Sigma</td>
			<td>D8537</td>
			<td>for MBMEC and immune cell isolation</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>Puromycin</td>
			<td>Sigma</td>
			<td>P8833</td>
			<td>for MBMEC culture medium; only for the first three days</td>
		</tr>
		<tr>
			<td>0.05% Trypsin-EDTA</td>
			<td>Gibco</td>
			<td>25300-054</td>
			<td>for harvesting MBMECs</td>
		</tr>
		<tr>
			<td>Collagenase Type IA</td>
			<td>Sigma</td>
			<td>C9891</td>
			<td>for dendritic cell isolation</td>
		</tr>
		<tr>
			<td>Trypan Blue solution, 0.4%</td>
			<td>Thermo Fisher Scientific</td>
			<td>15250061</td>
			<td>for cell counting</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma</td>
			<td>E5134</td>
			<td>for immune cell isolation</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>X-VIVO 15</td>
			<td>Lonza</td>
			<td>BE04-418Q</td>
			<td>protect from light; for B cell culture medium</td>
		</tr>
		<tr>
			<td>&#946;-mercaptoethanol</td>
			<td>Gibco</td>
			<td>31350-010</td>
			<td>for B cell culture medium</td>
		</tr>
		<tr>
			<td>L-Glutamine (100x Glutamax)</td>
			<td>Gibco</td>
			<td>35050-061</td>
			<td>for B cell culture medium</td>
		</tr>
		<tr>
			<td>mouse MOG35&#8212;55 peptide</td>
			<td>Biotrend</td>
			<td>BP0328</td>
			<td>for antigen-specific T cell activation</td>
		</tr>
		<tr>
			<td>purified anti-mouse CD3 Ab</td>
			<td>BioLegend</td>
			<td>100302</td>
			<td>clone 145-2C11; for polyclonal T cell activation</td>
		</tr>
		<tr>
			<td>purified NA/LE anti-mouse CD28 Ab</td>
			<td>BD Pharmingen</td>
			<td>553294</td>
			<td>clone 37.51; for polyclonal T cell activation</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>
	</tbody>
</table>

an in vitro model of the blood-brain barrier using impedance spectroscopy: a focus on t cell-endothelial cell interaction

Breakdown of the blood-brain barrier (BBB) is a critical step in the development of autoimmune diseases such as multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). This process is characterized by the transmigration of activated T cells across brain endothelial cells (ECs), the main constituents of the BBB. However, the consequences on brain EC function upon interaction with such T cells are largely unknown. Here we describe an assay that allows for the evaluation of primary mouse brain microvascular EC (MBMEC) function and barrier integrity during the interaction with T cells over time. The assay makes use of impedance cell spectroscopy, a powerful tool for studying EC monolayer integrity and permeability, by measuring changes in transendothelial electrical resistance (TEER) and cell layer capacitance (Ccl). In direct contact with ECs, stimulated but not na&#239;ve T cells are capable of inducing EC monolayer dysfunction, as visualized by a decrease in TEER and an increase in Ccl. The assay records changes in EC monolayer integrity in a continuous and automated fashion. It is sensitive enough to distinguish between different strengths of stimuli and levels of T cell activation and it enables the investigation of the consequences of a targeted modulation of T cell-EC interaction using a wide range of substances such as antibodies, pharmacological reagents and cytokines. The technique can also be used as a quality control for EC integrity in in vitro T-cell transmigration assays. These applications make it a versatile tool for studying BBB properties under physiological and pathophysiological conditions.

Figure 1 provides a general overview of the in vitro BBB model used to study the interaction between T cells and endothelial cells. The experiment consists of three major steps. The first step is the isolation of primary MBMECs from brain cortices, and their culture for five days. When they reach confluence in the cell culture plate, MBMECs are trypsinized and reseeded onto permeable inserts, which are then placed in the TEER instrument. The TEER and Ccl of MBMEC...

Watch this Scientific Journal Video about An In Vitro Model of the Blood-brain Barrier Using Impedance Spectroscopy: A Focus on T Cell-endothelial Cell Interaction at JoVE.com

An In Vitro Model of the Blood-brain Barrier Using Impedance Spectroscopy: A Focus on T Cell-endothelial Cell Interaction

Here, we describe an in vitro murine model of the blood-brain barrier that makes use of impedance cell spectroscopy, with a focus on the consequences on endothelial cell integrity and permeability upon interaction with activated T cells.

An In Vitro Model of the Blood-brain Barrier Using Impedance Spectroscopy: A Focus on T Cell-endothelial Cell Interaction

Breakdown of the blood-brain barrier (BBB) is a critical step in the development of autoimmune diseases such as multiple sclerosis (MS) and its animal ...

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Research

JoVE Journal

Immunology and Infection

11.1K Views.  University Hospital Münster. Here, we describe an in vitro murine model of the blood-brain barrier that makes use of impedance cell spectroscopy, with a focus on the consequences on endothelial cell integrity and permeability upon interaction with activated T cells.

Video: An In Vitro Model of the Blood-brain Barrier Using Impedance Spectroscopy: A Focus on T Cell-endothelial Cell Interaction

Retroviral Transduction of T-cell Receptors in Mouse T-cells

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen presenting cells (APCs)1. This recognition leads to the activation of T-cells and a series of functional outcomes (e.g. cytokine production, killing of the target cells). Understanding the functional role of TCRs is critical to harness the power of the immune system to treat a variety of immunology related diseases (e.g. cancer or autoimmunity).
It is convenient to study TCRs in mouse models, which can be accomplished in several ways. Making TCR transgenic mouse models is costly and time-consuming and currently there are only a limited number of them available2-4. Alternatively, mice with antigen-specific T-cells can be generated by bone marrow chimera. This method also takes several weeks and requires expertise5. Retroviral transduction of TCRs into in vitro activated mouse T-cells is a quick and relatively easy method to obtain T-cells of desired peptide-MHC specificity. Antigen-specific T-cells can be generated in one week and used in any downstream applications. Studying transduced T-cells also has direct application to human immunotherapy, as adoptive transfer of human T-cells transduced with antigen-specific TCRs is an emerging strategy for cancer treatment6.
Here we present a protocol to retrovirally transduce TCRs into in vitro activated mouse T-cells. Both human and mouse TCR genes can be used. Retroviruses carrying specific TCR genes are generated and used to infect mouse T-cells activated with anti-CD3 and anti-CD28 antibodies. After in vitro expansion, transduced T-cells are analyzed by flow cytometry.

We present a protocol to produce antigen-specific mouse T-cells using retroviral transduction

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

In vitro Biofilm Formation in an 8-well Chamber Slide

The chronic nature of many diseases is attributed to the formation of bacterial biofilms which are recalcitrant to traditional antibiotic therapy. Biofilms are community-associated bacteria attached to a surface and encased in a matrix. The role of the extracellular matrix is multifaceted, including facilitating nutrient acquisition, and offers significant protection against environmental stresses (e.g. host immune responses). In an effort to acquire a better understanding as to how the bacteria within a biofilm respond to environmental stresses we have used a protocol wherein we visualize bacterial biofilms which have formed in an 8-well chamber slide. The biofilms were stained with the BacLight Live/Dead stain and examined using a confocal microscope to characterize the relative biofilm size, and structure under varying incubation conditions. Z-stack images were collected via confocal microscopy and analyzed by COMSTAT. This protocol can be used to help elucidate the mechanism and kinetics by which biofilms form, as well as identify components that are important to biofilm structure and stability.

In vitro Biofilm Formation in an 8-well Chamber Slide

This article describes the procedure for the formation and visualization of a bacterial biofilm grown within an 8-well chamber slide

The chronic nature of many diseases is attributed to the formation of bacterial biofilms which are recalcitrant to traditional antibiotic therapy. ...

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological deficiencies. GVHD is caused by mature alloreactive T cells present in the bone marrow graft that are infused into the recipient and cause damage to host organs. However, in mice, T cells must be added to the bone marrow inoculum to cause GVHD. Although extensive work has been done to characterize T cell responses post transplant, bioluminescent imaging technology is a non-invasive method to monitor T cell trafficking patterns in vivo. 
Following lethal irradiation, recipient mice are transplanted with bone marrow cells and splenocytes from donor mice. T cell subsets from L2G85.B6 (transgenic mice that constitutively express luciferase) are included in the transplant. By only transplanting certain T cell subsets, one is able to track specific T cell subsets in vivo, and based on their location, develop hypotheses regarding the role of specific T cell subsets in promoting GVHD at various time points. At predetermined intervals post transplant, recipient mice are imaged using a Xenogen IVIS CCD camera. Light intensity can be quantified using Living Image software to generate a pseudo-color image based on photon intensity (red = high intensity, violet = low intensity). 
Between 4-7 days post transplant, recipient mice begin to show clinical signs of GVHD. Cooke et al.1 developed a scoring system to quantitate disease progression based on the recipient mice fur texture, skin integrity, activity, weight loss, and posture. Mice are scored daily, and euthanized when they become moribund. Recipient mice generally become moribund 20-30 days post transplant. 
Murine models are valuable tools for studying the immunology of GVHD. Selectively transplanting particular T cell subsets allows for careful identification of the roles each subset plays. Non-invasively tracking T cell responses in vivo adds another layer of value to murine GVHD models.

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Murine bone marrow transplantation is a widely used technique to study immunological mechanisms governing graft-versus-host disease in humans. The ability to monitor T cell trafficking patterns in vivo allows for detailed analysis of the development and perpetuation of T cell responses during graft-versus-host disease.

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological ...

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

T cell exhaustion is a major factor in failed pathogen clearance during chronic viral infections. Immunoregulatory pathways, such as PD-1 and IL-10, are upregulated upon this ongoing antigen exposure and contribute to loss of proliferation, reduced cytolytic function, and impaired cytokine production by CD4 and CD8 T cells. In the murine model of LCMV infection, administration of blocking antibodies against these two pathways augmented T cell responses. However, there is currently no in vitro assay to measure the impact of such blockade on cytokine secretion in cells from human samples. Our protocol and experimental approach enable us to accurately and efficiently quantify the restoration of cytokine production by HIV-specific CD4 T cells from HIV infected subjects.
Here, we depict an in vitro experimental design that enables measurements of cytokine secretion by HIV-specific CD4 T cells and their impact on other cell subsets. CD8 T cells were depleted from whole blood and remaining PBMCs were isolated via Ficoll separation method. CD8-depleted PBMCs were then incubated with blocking antibodies against PD-L1 and/or IL-10R&#945; and, after stimulation with an HIV-1 Gag peptide pool, cells were incubated at 37 &#176;C, 5% CO2. After 48 hr, supernatant was collected for cytokine analysis by beads arrays and cell pellets were collected for either phenotypic analysis using flow cytometry or transcriptional analysis using qRT-PCR. For more detailed analysis, different cell populations were obtained by selective subset depletion from PBMCs or by sorting using flow cytometry before being assessed in the same assays. These methods provide a highly sensitive and specific approach to determine the modulation of cytokine production by antigen-specific T-helper cells and to determine functional interactions between different populations of immune cells.

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

We developed an in vitro assay to investigate the role of immunoregulatory pathways in the regulation of cytokine secretion by HIV-specific CD4 T cells.

T cell exhaustion is a major factor in failed pathogen clearance during chronic viral infections. Immunoregulatory pathways, such as PD-1 and IL-10, are ...

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a prerequisite for pneumococcal infections such as pneumonia and otitis media. In vitro adherence assays can be used to study the attachment of pneumococci to epithelial cell monolayers and to investigate potential interventions, such as the use of probiotics, to inhibit pneumococcal&#160;colonization.&#160;The protocol described here is used to investigate the effects of the probiotic Streptococcus salivarius on the adherence of pneumococci to the human epithelial cell line CCL-23 (sometimes referred to as HEp-2 cells). The assay involves three main steps: 1) preparation of epithelial and bacterial cells, 2) addition of bacteria to epithelial cell monolayers, and 3) detection of adherent pneumococci by viable counts (serial dilution and plating) or quantitative real-time PCR (qPCR). This technique is relatively straightforward and does not require specialized equipment other than a tissue culture setup. The assay can be used to test other probiotic species and/or potential inhibitors of pneumococcal colonization and can be easily modified to address other scientific questions regarding pneumococcal adherence and invasion.

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

In vitro adherence assays can be used to study the attachment of Streptococcus pneumoniae to epithelial cell monolayers and to investigate potential interventions such as the use of probiotics for inhibiting pneumococcal colonization.

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a ...

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

Death of oligodendrocytes accompanied by destruction of neurons and axons are typical histopathological findings in cortical and subcortical grey matter lesions in inflammatory demyelinating disorders like multiple sclerosis (MS). In these disorders, mainly CD8+ T-cells of putative specificity for myelin- and oligodendrocyte-related antigens are found, so that neuronal apoptosis in grey matter lesions may be a collateral effect of these cells. Different types of animal models are established to study the underlying mechanisms of the mentioned pathophysiological processes. However, although they mimic some aspects of MS, it is impossible to dissect the exact mechanism and time course of &#8216;&#8216;collateral&#8217;&#8217; neuronal cell death. To address this course, here we show a protocol to study the mechanisms and time response of neuronal damage following an oligodendrocyte-directed CD8+ T cell attack. To target only the myelin sheath and the oligodendrocytes, in vitro activated oligodendrocyte-specific CD8+ T-cells are transferred into acutely isolated brain slices. After a defined incubation period, myelin and neuronal damage can be analysed in different regions of interest. Potential applications and limitations of this model will be discussed.

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

To address mechanisms of demyelination and neuronal apoptosis in cortical lesions of inflammatory demyelinating disorders, different animal models are used. We here describe an ex vivo approach by using oligodendrocyte-specific CD8+ T-cells on brain slices, resulting in oligodendroglial and neuronal death. Potential applications and limitations of the model are discussed.

Death of oligodendrocytes accompanied by destruction of neurons and axons are typical histopathological findings in cortical and subcortical grey matter ...

Development of an in vitro model system for studying the interaction of Equus caballus IgE with its high-affinity receptor Fc&#949;RI

The interaction of IgE with its high-affinity Fc receptor (Fc&#949;RI) followed by an antigenic challenge is the principal pathway in IgE mediated allergic reactions. As a consequence of the high affinity binding between IgE and Fc&#949;RI, along with the continuous production of IgE by B cells, allergies usually persist throughout life, with currently no permanent cure available. Horses, especially race horses, which are commonly inbred, are a species of mammals that are very prone to the development of hypersensitivity responses, which can seriously affect their performance. Physiological responses to allergic sensitization in horses mirror that observed in humans and dogs. In this paper we describe the development of an in situ assay system for the quantitative assessment of the release of mediators of the allergic response pertaining to the equine system. To this end, the gene encoding equine Fc&#949;RI&#945; was transfected into and expressed onto the surface of parental Rat Basophil Leukemia (RBL-2H3.1) cells. The gene product of the transfected equine &#945;-chain formed a functional receptor complex with the endogenous rat &#946;- and &#947;-chains 1. The resultant assay system facilitated an assessment of the quantity of mediator secreted from equine Fc&#949;RI&#945; transfected RBL-2H3.1 cells following sensitization with equine IgE and antigenic challenge using &#946;-hexosaminidase release as a readout 2, 3. Mediator release peaked at 36.68% &#177; 4.88% at 100 ng ml-1 of antigen. This assay was modified from previous assays used to study human and canine allergic responses 4, 5. We have also shown that this type of assay system has multiple applications for the development of diagnostic tools and the safety assessment of potential therapeutic intervention strategies in allergic disease 6, 2, 3.

The current study describes the development and applications of a genetically engineered assay system based on the transfection of rat basophilic leukemia cells with the equine Fc&#949;RI&#945; gene. Transfected cells express a functional receptor where the release of mediators of the allergic response can be activated by IgE and antigen.

The interaction of IgE with its high-affinity Fc receptor (Fc&#949;RI) followed by an antigenic challenge is the principal pathway in IgE mediated ...

Mouse Na&#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition of cognate antigen presented on MHC class II. The cytokine signals present in the environment at the time of TCR activation are a major factor in determining the effector fate of a na&#239;ve CD4+ T cell. Although the cytokine environment during na&#239;ve T cell activation may be complex and involve both redundant and opposing signals in vivo, the addition of various cytokine combinations during naive CD4+ T cell activation in vitro can readily promote the establishment of effector T helper lineages with hallmark cytokine and transcription factor expression. Such differentiation experiments are commonly used as a first step for the evaluation of targets believed to promote or inhibit the development of certain CD4+ T helper subsets. The addition of mediators, such as signaling agonists, antagonists, or other cytokines, during the differentiation process can also be used to study the influence of a particular target on T cell differentiation. Here, we describe a basic protocol for the isolation of na&#239;ve T cells from mouse and the subsequent steps necessary for polarizing na&#239;ve cells to various T helper effector lineages in vitro.

Mouse Na&#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Na&#239;ve CD4+ T cells polarize to various subsets depending on the environment at the time of activation. The differentiation of na&#239;ve CD4+ T cells to various effector subsets can be achieved in vitro through the addition of T cell receptor stimuli and specific cytokine signals.

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition ...

Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the development and persistence of many health related infections. One of the most well described and studied biofilms in human disease occurs in chronic pulmonary infection of cystic fibrosis patients. When studying biofilms in the context of the host, many factors can impact biofilm formation and development. In order to identify how host factors may affect biofilm formation and development, we used a static chambered coverglass method to grow biofilms in the presence of host-derived factors in the form of sputum supernatants. Bacteria are seeded into chambers and exposed to sputum filtrates. Following 48 hr of growth, biofilms are stained with a commercial biofilm viability kit prior to confocal microscopy and analysis. Following image acquisition, biofilm properties can be assessed using different software platforms. This method allows us to visualize key properties of biofilm growth in presence of different substances including antibiotics.

This protocol describes the visualization of biofilm development following exposure to host-factors using a slide chamber model. This model allows for direct visualization of biofilm development as well as analysis of biofilm parameters using computer software programs.

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the ...

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte extravasation might result in pathophysiological changes in the CNS. Thus, investigation of lymphocyte migration into the CNS is important to understand inflammatory CNS diseases and to develop new therapy approaches. Here we present an in vitro model of the human blood-brain barrier to study lymphocyte extravasation. Human brain microvascular endothelial cells (HBMEC) are confluently grown on a porous polyethylene terephthalate transwell insert to mimic the endothelium of the blood-brain barrier. Barrier function is validated by zonula occludens immunohistochemistry, transendothelial electrical resistance (TEER) measurements as well as analysis of evans blue permeation. This model allows investigation of the diapedesis of rare lymphocyte subsets such as CD56brightCD16dim/- NK cells. Furthermore, the effects of other cells, cytokines and chemokines, disease-related alterations, and distinct treatment regimens on the migratory capacity of lymphocytes can be studied. Finally, the impact of inflammatory stimuli as well as different treatment regimens on the endothelial barrier can be analyzed.

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Here, we describe a human blood-brain barrier model enabling to investigate lymphocyte transmigration into the central nervous system in vitro.