Name: Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier (Video) | JoVE Journal
Uploaded: 2017-04-05T14:00:00.000+00:00
Duration: 9 min
Description: Learn lymphocyte extravasation analysis using an in vitro human blood-brain barrier model. This method enables single-cell study of rare immune populations and advances understanding of CNS inflammatory diseases.

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5 minutes

This study has been supported by the Collaborative Research Centre CRC TR128 "Initiating/Effector versus Regulatory Mechanisms in Multiple Sclerosis-Progress towards Tackling the Disease" (Project A9 to H.W. and C.C.G., project B1 to N.S.).

1. Cell Culture of Human Brain Microvascular Endothelial Cells (HBMEC)
<ol>
	<li>Coating of cell culture flasks
	<ol>
		<li>To prepare the fibronectin solution, add 10 mL PBS to a 15 mL centrifuge tube. Add 150 µL fibronectin and mix well.</li>
		<li>To cover the bottom a T-25 cell culture flask add 2 mL of the fibronectin solution. Incubate the cell culture flask for at least 3 h at 37 °C in the incubator. Fibronectin coated flasks can be stored for 2 weeks at 37 °C / 5% CO2.</li>
	</ol>
	</li>
	<li>Seeding and cell culture of HBMEC
	<ol>
		<li>Aspirate fibronectin solution from the bottom of the cell culture flask. Add 7.2 x 104 HBMEC/cm² suspended in 6 mL ECM-b medium (= ECM-b supplemented with 5% fetal bovine serum, 1% penicillin/streptomycin, and 1% endothelial cell growth supplement). Incubate at 37 °C / 5% CO2. Check cell growth daily using a microscope.</li>
		<li>Change the medium every 3 days. Harvest or split cells, when HBMEC reach approximately 80% confluence. HBMEC should be used between passage 1 and 15 to avoid loss of physiological properties.</li>
	</ol>
	</li>
	<li>Harvest HBMEC.
	<ol>
		<li>Prepare accutase solution by mixing accutase (1x) with PBS at a ratio of 1:1. Keep accutase solution at 37 °C in a water bath until further use.</li>
		<li>Transfer ECM-b medium from the cell culture flask to a 15 mL centrifuge tube. Wash HBMEC by adding 5 mL PBS to the bottom of the cell culture flask. Aspirate PBS and repeat the washing step two more times.</li>
		<li>Add 2 mL pre-warmed accutase solution. Incubate at 37 °C for 2 min. Afterwards, HBMEC are re-suspended by firmly tapping the cell culture flask several times. Cell detachment is controlled using a microscope</li>
		<li>The ECM-b-medium previously stored in a 15 mL tube is added back to the cell culture flask as soon as HBMEC start to detach. Rinse the bottom of the flask repeatedly until most HBMEC are re-suspended.</li>
		<li>Transfer the cell suspension to a 15 mL centrifuge tube. Centrifuge at 300 x g for 10 min at room temperature. Discard supernatant and re-suspend cells in 1 mL ECM-b medium. Count cells and dilute the cell suspension to achieve a final concentration of 3 x 105 HBMEC per mL ECM-b medium.</li>
	</ol>
	</li>
</ol>
2. Preparation of the Cell Culture Inserts
<ol>
	<li>Coating of cell culture inserts 
	Important note: Avoid touching the membrane of the cell culture inserts.
	<ol>
		<li>Add 100 µL fibronectin solution (see 1.1.1) to each cell culture insert (Figure 1A) and one well of a 96-well flat bottom plate (optical control well). Incubate for at least 3 h at 37 °C. After incubation aspirate fibronectin solution.</li>
		<li>Add 100 µL HBMEC suspension to the cell culture inserts and the optical control well. Add 600 µl ECM-b medium to the lower compartment of the cell culture inserts. Incubate for 3 - 4 days at 37 °C / 5% CO2 until barrier integrity (Figure 1B) is reached, check cell growth by microscopic evaluation of the HBMEC in the optical control well. Note: Cell growth beyond four days is not recommended.</li>
		<li>Optional: To mimic inflammatory conditions aspirate the medium from the lower compartment and replace it with ECM-b medium supplemented with 500 U/mL IFN-γ/TNF-α 24 h prior the migration assay.</li>
	</ol>
	</li>
</ol>
3. Quality Control with Evans Blue on the Day of the Transmigration Assay
<ol>
	<li>Preparation of evans blue solution
	<ol>
		<li>To prepare PBS/B27 solution mix 10 mL PBS with 200 µl B27 supplement using a 15 mL centrifuge tube. Dilute evans blue stock solution (20 mg/mL PBS) 1:1,000 with PBS/B27.</li>
	</ol>
	</li>
	<li>Evans blue permeability assay
	<ol>
		<li>Aspirate the medium from the lower compartment followed by the upper compartment of one cell culture insert containing a confluent HBMEC monolayer. Add 100 µL evans blue solution to the cell culture insert.</li>
		<li>Add 600 µL PBS/B27 to the lower compartment and incubate for 60 min at 37 °C / 5% CO2. Carefully remove the cell culture insert using forceps.</li>
	</ol>
	</li>
	<li>Evans blue measurement
	<ol>
		<li>Remove PBS/B27 from the lower compartment and transfer 100 µL each to two wells of a black polystyrol 96-well flat bottom plate. Insert plate in a Tecan Infinite M200 Pro plate reader and determine optimal z-position.</li>
		<li>Measure excitation of evans blue using respective settings (for example: excitation: 620 nm, emission: 680 nm, excitation bandwidth: 9 nm, emission bandwidth: 20 nm, 175x enhancement, 25 flashes, time of integration: 20 µs).</li>
		<li>To determine HBMEC barrier functions compare acquired data to a standard curve depicting evans blue permeation across HBMEC at different time points after seeding cells (Figure 1B, right).</li>
	</ol>
	</li>
</ol>
4. Migration Assay
<ol>
	<li>Preparation of peripheral blood mononuclear cells (PBMC).
	<ol>
		<li>Add 10 mL RPMI into a 15 mL centrifuge tube and add 200 µL B27 supplement. Count PBMC and centrifuge cells at 300 x g for 5 min. Re-suspend PBMC to a final concentration of 5 x 106 cells/mL RPMI/B27.</li>
	</ol>
	</li>
	<li>Set-up of the migration assay
	<ol>
		<li>Aspirate medium from the lower compartment followed by the upper compartment of cell culture inserts containing confluent HBMEC monolayers (Figure 1A). Per donor add 100 µL PBMC suspension each to the cell culture inserts and also to one well of a 24-well plate per (in vitro control).</li>
		<li>Add 600 µL RPMI/B27 to the lower compartment of the cell culture inserts and 500 µL to the PBMC of the in vitro control and incubate 6 h at 37 °C / 5% CO2.</li>
	</ol>
	</li>
	<li>Harvesting of migrated PBMC
	<ol>
		<li>Take out the cell culture insert using forceps and carefully rinse the bottom with 400 µL PBS without touching the membrane. Discard the cell culture insert.</li>
		<li>Add 20 µL flow count fluorospheres (approximately 1,000 beads/µL) to the lower compartment of the cell culture insert as well as to the in vitro control and mix well. Transfer 1 mL of resulting PBMC suspension to flow cytometry tubes.</li>
	</ol>
	</li>
</ol>
5. Flow Cytometry
<ol>
	<li>Sample preparation
	<ol>
		<li>Centrifuge PBMC at 300 x g for 5 min at room temperature.</li>
		<li>Prepare antibody solution by adding fluorochrome-conjugated antibodies to 100 µL flow cytometry buffer (PBS/1% BSA/2 mM EDTA) per sample. For the results presented below 1 µL CD4-FITC, 1 µLCD3-PerCP/Cy5.5, 1 µL CD56-PC7, 1 µL CD8-A700, and 1 µL CD16-A750 were used per sample.</li>
		<li>Re-suspend PBMC in 100 µL of the antibody solution and incubate for 30 min at 4 °C.</li>
		<li>Add 250 µL flow cytometry buffer and centrifuge at 300 x g for 5 min.</li>
	</ol>
	</li>
	<li>Sample acquisition
	<ol>
		<li>Re-suspend PBMC in the required amount (varies depending on the flow cytometer used) of flow cytometry buffer.</li>
		<li>Acquire stained PBMC using a flow cytometer with an active detector between 525 and 700 nm wavelength to detect flow count fluorospheres (excitation 488 nm, emission 525 - 700 nm). 
		(The following steps are an example if a Gallios flow cytometer operated with Kaluza G software is used: (1) Start the computer. (2) When the operating system is fully loaded, start the flow cytometer by pressing the "cytometer on" button. (3) Load the respective acquisition protocol by pressing "open protocol" button. (4) Choose the required protocol and select "open". (5) Duplicate the protocol for every sample by clicking with the right mouse button on the protocol visible in the virtual carousel and a left click on the field "duplicate". (6) Label each sample in the sample list. (7) Transfer the samples to the indicated positions of the carousel and start the acquisition.)</li>
	</ol>
	</li>
	<li>Sample analysis
	<ol>
		<li>Open resulting flow cytometry data using the respective software. Determine number of subpopulations of interest for transmigrated PBMC as well as cells from in vitro control wells and flow count fluorospheres using the respective analysis software. 
		(An example of the gating strategy is given in the results part (Figure 1 C: To analyze the transmigration of NK-cell subsets, first select lymphocytes in a sideward scatter channel (SSC) versus forward scatter channel (FSC) plot. Lymphocytes are then displayed in a CD3 versus CD56 plot and CD56+CD3- NK cells are selected. To distinguish between NK-cell subsets, NK cells are displayed in a CD56 versus CD16 plot and CD56brightCD16dim/- as well as CD56dimCD16+ NK cells are selected. In addition, flow count fluorospheres are selected from a FSC versus SSC plot and subsequently displayed in a plot of a channel with an emission between 525 and 700 nm versus time to determine their number.)</li>
		<li>To calculate the total cell number of each sample, normalize the detected number of cells using flow count fluorospheres: 
		<img alt="Cell quantification formula; equation for cell count using flow cytometry, fluorosphere reference." src="/files/ftp_upload/55390/55390eq1.jpg" data-altupdatedat="1788250015000" data-alt="Equation"></li>
		<li>Determine percentage of migrated cells as ratio between total migrated cells and total cells in the in vitro control.</li>
	</ol>
	</li>
</ol>

The author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: A.S.-M. and U.B. have no financial disclosures. T. S.-H. received travel and conference expenses from Biogen. N.S. received speaker and advisory board honoraria from Biogen and Novartis Pharma, as well as travel expenses from Biogen. H.W. received compensation for serving on Scientific Advisory Boards/Steering Committees for Bayer Healthcare, Biogen, Merck Serono, Novartis, and Sanofi-Genzyme. He also received speaker honoraria and travel support from Bayer Vital GmbH, Bayer Schering AG, Biogen, CSL Behring, Fresenius Medical Care, Glaxo Smith Kline, GW Pharmaceuticals, Lundbeck, Merck Serono, Omniamed, Novartis, and Sanofi-Genzyme. He received compensation as a consultant from Biogen, Merck Serono, Novartis, and Sanofi-Genzyme. H.W. received research support from Bayer Vital, Biogen, Genzyme, Merck Serono, Novartis, Sanofi-Aventis Germany, and Sanofi US. C.C.G. received speaker honoraria and travel expenses for attending meetings from Genzyme, Novartis Pharma GmbH, and Bayer Health Care.

Here we present a technique to investigate the transmigration of lymphocytes across the human blood-brain barrier. In vitro analysis of lymphocyte migration to the CNS is important to study basic processes of lymphocyte extravasation, potential disease-related alterations, and new therapeutic approaches.
Several modifications of the blood-brain barrier model are possible. For example, cells from the upper compartment could be analyzed to investigate the composition of the non-migrated...

Lymphocyte migration from the blood into tissues is crucial for immune surveillance. A sequence of specific molecular interactions ensures site specific extravasation into small intestine, skin, lymph nodes, the central nervous system (CNS), and other tissues1. Alterations in lymphocyte migration are involved in the pathophysiology of a number of wide spread diseases2. Migration into the immune-privileged CNS is tightly regulated and accordingly alterations of this process are involved in CNS-related diseases like encephalomyelitis3, neuromyelitis optica, stroke, and multiple sclerosis (MS)2,4,5,6,7. Therefore, it is important to study lymphocyte extravasation to better understand disease pathophysiology and to develop tools for amelioration of disease burden8,9,10,11,12.
Lymphocytes migrate into the CNS via distinct routes. Extravasation through postcapillary venules into the subarachnoid space via the blood-cerebrospinal fluid barrier within the choroid plexus and across the blood-brain barrier have been described1,13,14,15. Migration across the blood-brain barrier is conducted by the interaction of lymphocytes with endothelial cells14. In contrast to endothelial cells in the periphery, endothelial cells of the CNS express high amounts of tight junction molecules, thereby strictly limiting the amount of cells and proteins capable of crossing the blood-brain barrier16. Inflammation results in loosening of tight junctions and induces the expression of adhesion molecules; thus, enhancing lymphocyte migration into the CNS1,17,18.
Extravasation via the blood-brain barrier is a multistep process. Lymphocytes tether to the endothelial cells and then roll along the endothelium in a process mainly mediated by selectins1,15. Subsequently, interactions between chemokines secreted by the endothelium and the respective chemokine receptors expressed on lymphocytes induce conformational changes of integrins, thereby promoting firm adhesion to the endothelial cells1. Finally, lymphocytes either crawl along the endothelial barrier against the blood flow before transmigrating into the perivascular space, or stall immediately and directly transmigrate at the site of firm adhesion1,19,20. All these steps of lymphocyte extravasation can be analyzed in vitro using distinct techniques21. Time-lapse video microscopy is used to study the initial tethering and rolling15. Adhesion assays provide detailed information about firm arrest to endothelial barriers22. Transmigration assays as demonstrated here allow analysis of immune-cell transmigration21,23,24,25,26,27,28,29.
Using the human in vitro blood brain barrier model, we could recently show that a higher migratory capacity of CD56brightCD16dim/- NK cells compared to their CD56dimCD16+ counterparts was reflected by a predominance of this NK cell subset in the intrathecal compartment21. Thus, our experimental setup seems to be suitable to mimic the in vivo situation.

<table><tbody><tr><td>PBS</td><td>Gibco</td><td>14190-094</td><td>without CaCl&lt;sub&gt;2&lt;/sub&gt; or MgCl&lt;sub&gt;2&lt;/sub&gt;</td></tr><tr><td>Fibronectin 1 mg/mL</td><td>Sigma</td><td>F1141-5MG</td><td>from bovine plasma</td></tr><tr><td>T-25 cell culture flask</td><td>Greiner BioOne</td><td>690160</td><td></td></tr><tr><td>HBMEC</td><td>ScienCell</td><td>1000</td><td></td></tr><tr><td></td><td>Pelobiotech</td><td>PB-H-6023</td><td></td></tr><tr><td>Accutase</td><td>Sigma</td><td>A6964-100ML</td><td></td></tr><tr><td>ECM-b</td><td>ScienCell</td><td>1001-b</td><td></td></tr><tr><td>FBS</td><td>ScienCell</td><td>1001-b</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>ScienCell</td><td>1001-b</td><td></td></tr><tr><td>Endothelial cell growth supplement</td><td>ScienCell</td><td>1001-b</td><td></td></tr><tr><td>Transwell</td><td>Corning</td><td>3472</td><td>clear, 6.5 mm diameter, 3.0 &amp;micro;m pore size</td></tr><tr><td>96-well flat bottom plate</td><td>Corning</td><td>3596</td><td></td></tr><tr><td>Evans blue</td><td>Sigma</td><td>E2129-10G</td><td>stock solution: 1 g/50 mL PBS</td></tr><tr><td>B27</td><td>Gibco</td><td>17504-044</td><td>50x concentrated</td></tr><tr><td>Infinite M200Pro</td><td>Tecan</td><td></td><td></td></tr><tr><td>96-well black flat bottom plate</td><td>Greiner BioOne</td><td>675086</td><td></td></tr><tr><td>48-well plate</td><td>Corning</td><td>3526</td><td></td></tr><tr><td>RPMI 1640</td><td>Gibco</td><td>61870-010</td><td></td></tr><tr><td>Flow Count Fluorospheres</td><td>Beckman Coulter</td><td>7547053</td><td></td></tr><tr><td>Na-EDTA</td><td>Sigma</td><td>E5134</td><td></td></tr><tr><td>BSA</td><td>Sigma</td><td>A2153</td><td></td></tr><tr><td>Gallios 10-color flow cytometer</td><td>Beckman Coulter</td><td></td><td></td></tr><tr><td>Kaluza 1.5a</td><td>Beckman Coulter</td><td></td><td></td></tr><tr><td>TNF-&amp;alpha;</td><td>Peprotech</td><td>300-01A</td><td></td></tr><tr><td>IFN-&amp;gamma;</td><td>Peprotech</td><td>300-02</td><td></td></tr><tr><td>CD3-PerCP/Cy5.5</td><td>Biolegend</td><td>300430</td><td>clone UCHT1</td></tr><tr><td>CD56-PC7</td><td>Beckman Coulter</td><td>A21692</td><td>clone N901</td></tr><tr><td>CD16-A750</td><td>Beckman Coulter</td><td>A66330</td><td>clone 3G8</td></tr><tr><td>CD4-FITC</td><td>Biolegend</td><td>300506</td><td>clone RPA-T4</td></tr><tr><td>CD8-A700</td><td>Beckman Coulter</td><td>A66332</td><td>clone B9.11</td></tr></tbody></table>

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 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.

Representative results showing transmigration of NK-cell and T-cell subsets using the human blood-brain barrier model (Figure 1A) are shown. The integrity of the HBMEC monolayer was validated by staining of the tight junction molecule ZO-1, transendothelial electrical resistance (TEER) measurements, and evans blue permeation (Figure 1B). Following 3 - 4 days culture HBMEC expressed the tight junction molecule ZO-1 (Figure 1B, left). Furth...

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

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte ...

The overall goal of this transmigration assay is to investigate lymphocyte extravasation into the central nervous system in vitro. This method can help in furthering our understanding of inflammatory diseases of the central nervous system and in the development of new therapeutic approaches. Some advantages of this technique are that it is easily accessible and that it allows analysis of rare lymphocyte populations on single cell level.This method can provide insight into lymphocyte transmigration across the blood-brain barrier and can be applied to the study of transmigration via the blood-cerebrospinal fluid barrier. In addition, Evans blue permeation analysis can be performed to assess the integrity of the blood-brain barrier model. Begin by covering the bottom of a T-25 cell culture flask with two milliliters of freshly prepared fibronectin solution for an at least three-hour incubation in a cell culture incubator at 37 degrees Celsius and 5%carbon dioxide.At the end of the incubation, replace the fibronectin solution with 7.2 times 10 to the fourth human brain microvascular endothelial cells, or HBMEC, in six milliliters of complete ECM-b medium, and return the flask to the incubator. When the HBMEC reach an 80%confluency, transfer the supernatant to a 15-milliliter conical tube, and wash the adherent cells three times with five milliliters of PBS. After the third wash, add two milliliters of pre-warmed Accutase solution to the cells for two minutes at 37 degrees Celsius.Then firmly tap the flask several times, confirming the cell detachment by light microscopy. When the cells begin to lift from the bottom of the flask, use the harvested supernatant to rinse the flask repeatedly until most of the HBMEC are resuspended. Then transfer the cell suspension to a 15-milliliter conical tube for centrifugation, and resuspend the pellet in one milliliter of fresh ECM-b medium for counting.To prepare the cell culture inserts for a transmigration assay, first add 100 microliters of fibronectin solution to each insert and to one well of a 96-well flat bottom plate. After three hours at 37 degrees Celsius, replace the fibronectin solution with 100 microliters of HBMEC, and add 600 microliters of fresh ECM-b medium to the lower compartments of the cell culture inserts. Then return the plates to the cell culture incubator for three to four days.To test whether barrier integrity has been reached, aspirate the medium from the lower and upper compartments of one cell culture insert, and add 100 microliters of freshly prepared Evans blue solution to the insert. Add 600 microliters of PBS supplemented with B27 to the lower compartment, and return the plate to the incubator. After one hour, use forceps to carefully remove the insert, and transfer 100 microliters of PBS B27 from the lower compartment into two individual wells of a black, polystyrol, 96-well flat bottom plate.Then load the plate into a multi-mode microplate reader, and determine the optimal z-position. To determine the HBMEC barrier function, measure the excitation of the Evans blue dye solution using the appropriate settings, and compare the acquired data to a standard curve depicting Evans blue permeation across the HBMEC at different time points after seeding. After confirming the formation of the cell monolayer, resuspend the peripheral blood mononuclear cell, or PBMC, sample to a five times 10 to the sixth cells per milliliter concentration in RPMI medium supplemented with B27.Next, aspirate the medium from the lower and upper compartments of the appropriate number of cell culture inserts containing the confluent HBMEC monolayers, and add 100 microliters of PBMC per donor to the cell culture inserts and to one well of a 24-well plate per in vitro control. Add 600 microliters of RPMI plus B27 to the lower compartments of the cell culture inserts and 500 microliters to the in vitro control wells, and return the plates to the cell culture incubator for six hours. At the end of the incubation, use forceps to remove the cell culture inserts, carefully rinsing the bottoms with 400 microliters of PBS without touching the membranes.Then thoroughly mix 20 microliters of Flow-Count Fluorospheres with the cells in the experimental and in vitro control wells, and transfer one milliliter of the resulting PBMC suspensions to the appropriate corresponding flow cytometry tubes. Collect the cells by centrifugation, and add the fluorochrome-conjugated antibodies of interest in 100 microliters of flow cytometry buffer to each sample. After 30 minutes at four degrees Celsius, wash the cells with 250 microliters of fresh flow cytometry buffer, and resuspend the pellets in the appropriate volume of flow cytometry buffer for analysis.When all of the data have been acquired, open the appropriate flow cytometry analysis software. To analyze the NK cell subset transmigration, first select the lymphocytes in a side versus forward scatter channel plot, and display the cells in a CD3 versus CD56 dot plot. Then select the CD56-positive, CD3-negative NK cells.To distinguish between the transmigrated NK cell subsets, display the cells in a CD56 versus CD16 plot and gate for the CD56bright, CD16dim or negative, and CD56dim, CD16-positive cells. Then select the Flow-Count Fluorospheres from a forward versus side scatter plot. To determine the bead number, display the selected data in a plot of a channel covering an emission wavelength between 525 and 700 nanometers versus time.Finally, determine the percentage of migrated NK cells as the ratio between the total number of migrated cells and the total number of cells in the in vitro control. Following three to four days of culture, the HBMEC expressed the tight junction molecule Zonula occludens-1 and grow in monolayers that exhibit transendothelial electrical resistance and reduced Evans blue dye permeation. Stimulation of the endothelial cell monolayer with interferon gamma and TNF alpha for 24 hours prior to a whole PBMC transmigration assay results in an increased migration of all of the analyzed lymphocyte populations.Interestingly, CD56bright, CD16dim or negative NK cells exhibit a higher migratory capacity than CD56dim, CD16-positive NK across both unstimulated and cytokine-activated HBMEC. Although the relative increase of transmigration through an inflamed cell monolayer is higher for the CD56dim cells overall, mimicking the in vivo observations that CD56bright, CD16dim-negative NK cells are enriched in the intrathecal compartment. One mastered, this technique can be completed within 10 hours.Pretreatment of the in vitro transmigration setup with interferon gamma and TNF alpha enables the study of lymphocyte migration under inflammatory conditions. Our protocol may be modified by applying shear forces or by co-cultivation with other cells of the blood-brain barrier, like astrocytes, to provide a refined model for lymphocyte transmigration into the central nervous system. After watching this video, you should have a good understanding of how to investigate lymphocyte transmigration using an in vitro model of the human blood-brain barrier.

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JoVE Journal

Immunology and Infection

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

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