Name: an in vitro model of the blood-brain barrier using impedance spectroscopy: a focus on t cell-endothelial cell interaction
Uploaded: 2016-12-08T12:30:00
Description: 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

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 wi...

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 ...

The overall goal of this experiment is to study the consequences on endothelial cell integrity and permeability upon interaction with activated T cells. This method helps to answer key questions in the neuroimmunology field, such as how does the direct interaction between T cells and endothelial cells affect blood-brain barrier integrity and permeability. The main advantage of this technique is that it enables automated, real-time assessment of barrier function upon targeted modulation of T cell and endothelial cell interaction.The implications of this technique extend toward therapy of autoimmune CNS disorders, because it provides insight into T cell mediated blood-brain barrier breakdown, a crucial step into pathogenesis of such diseases. This method focuses on marine blood-brain barrier properties during EAE. But it can also be applied to red or human blood-brain barrier models, as well as studies of other autoimmune CNS disorders.Generally, individuals new to this method may struggle because of possible difficulties with optimal timing of combined primary endothelial cell culture and T cell activation. We first developed this method when we set off to investigate effects of different T cell activation levels on the blood-brain barrier properties during EAE, an animal model of multiple sclerosis. Visual demonstration of this method is critical, as setting up T cell and endothelial cell co-cultures is difficult to learn, because initial variations in T between wells may need to subsequent misrepresentations of the results.To begin this procedure, on day five after MBMEC isolation, precoat the permeable inserts with 80 microliters of MBMEC coating solution per insert. Incubate them at 37 degrees Celsius, and 5%carbon dioxide, for three hours. After removing the MBMEC coating solution from inserts, wash MBMECs by adding two milliliters of room temperature PBS to each well, and aspirating it after one minute, and then repeat this procedure once more.Next, add 0.05%Trypsin EDTA at 300 microliters per well and incubate it for five to ten minutes. Subsequently, add two milliliters of MBMEC medium to each well to stop trypsinization. Then, transfer the collected cells to a 15 milliliter centrifugation tube and centrifuge the sample at 700 times g for eight minutes at four degrees Celsius.After eight minutes, discard the supernatant and resuspend the cells in one milliliter of MBMEC medium without puromycin. To count the cells, mix 90 microliters of 0.04%trypan blue. With 10 microliters of cell suspension, pipette 10 microliters of the mixture between the glass cover and the hemocytometer.Next, cede two times 10 to the fourth cells per insert. Add 810 microliters of MBMEC medium to the lower compartment of each well and place the plate with the inserts in the incubator until ready to proceed with TEER measurement. To stimulate CD4 positive T cells, precoat a round-bottomed 96 well plate with purified anti-mouse CD3 antibody at a desired final concentration for three hours at 37 degree Celsius.After isolating the T cells, wash the precoated plate twice with PBS. Next, add purified anti-CD28 antibody to the isolated T cells at a desired final concentration and mix well. Then, cede the T cells and leave them at 37 degrees Celsius for two to three days.To set up TEER measurement, place the 24 well module of the TEER instrument in the laminar flow hood. Remove the lids and place the inserts with MBMECs in the instrument, using forceps. Next, pipette 810 microliters of the fresh medium to the lower compartment of the module wells, added carefully between the insert and the wall of the module well.After that, close the lids and place the instrument in the incubator. Connect the instrument to the computer. Then, turn on the instrument controller and open the software.In the pop-up window, select New Measurement"Check show TER"and show Ccl"boxes. Then, press start. After completing the first measurement, select check all wells"in the Results tab to see all TEER and Ccl values.Then, save the file. Next, choose the time point to co-culture MBMECs with T cells when Ccl is stable and lower than one microfarad per square centimeter and TEER has reached its maximum level. Carefully inspect the absolute TEER and Ccl values and exclude the wells in which MBMECs have not developed confluent enough monolayers by unchecking such wells.Group the rest of the wells by right-clicking on the wells and selecting add well to new average well"Name it in the pop-up window and do the same for all individual wells to be grouped. Then check all average wells to confirm that all of them have the same initial conditions before the co-culture. In the experiment tab, press pause.Disconnect the instrument and take it out of the incubator. Then, remove the lids in the laminar flow hood. Subsequently, remove some of the medium from the insert at the upper well compartment.Add T cells to MBMECs by carefully pipetting 150 microliters of medium. Then, reconnect the instrument and press resume measurement. After 24 hours, press stop in the experiment tab and save the file.Shown here are the spindle-shaped morphology in the immunofluorescent staining of PECAM-1 and Cldn-5 five days after MBMEC isolation. Here are the TEER and Ccl values before and after grouping of individual wells prior to the addition of T cells. The blue curve is not used for the experiment since MBMECs in this well have not developed TEER as high as in other wells.In this experiment, mark 35 to 55 specific CD4 positive T cells were stimulated in an antigen-specific fashion for five days. Naive T cells and proinflammatory cytokines, interferon gamma and TNF-alpha served as a negative and positive control, respectively. Here, T cells were stimulated by the same amount of anti-CD3 and anti-CD28 antibodies and the ones cultured for three days exhibited a greater propensity for barrier disruption compared to the T cells cultured for only two days.The results here show that stimulated T cells but not their supernatants caused a substantial damage to the MBMECs, indicating the direct contact between T cells and MBMECs is critical for barrier disruption. On the other hand, adding granzyme b inhibitor two to T cell culture restored MBMEC integrity to a greater extent, pointing to the cytolytic molecule as an important player in causing MBMEC damage and subsequent decrease in TEER Once mastered, this experiment can be done in 12 days if it's performed properly. While attempting this procedure, it's important to remember to choose the correct time point for setting up the co-culture of T cells and endothelial cells.Following this procedure other essays can be employed, such as immunofluorescence microscopy, flow cytometry, and probability essays. In order to address additional questions, such as How does this interaction affect tight junction molecule expression permeability and the extent of T cell transmigration? After its development, this technique paved a way for researchers in the field of neuroimmunology to explore autoimmune disorders of the CNS in mice.After watching this video, you should have a good idea of how to assess the direct effects between activated T cells and endothelial cells with regard to blood-brain barrier integrity and permeability.

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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

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 ...

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 ...

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

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 ...

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 ...

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

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 ...

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol ...