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 &#181;L 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 &#176;C in the incubator. Fibronectin coated flasks can be stored for 2 weeks at 37 &#176;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/cm&#178; 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 &#176;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 &#176;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 &#176;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 &#181;L 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 &#176;C. After incubation aspirate fibronectin solution.</li>
		<li>Add 100 &#181;L HBMEC suspension to the cell culture inserts and the optical control well. Add 600 &#181;l ECM-b medium to the lower compartment of the cell culture inserts. Incubate for 3 - 4 days at 37 &#176;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-&#947;/TNF-&#945; 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 &#181;l 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 &#181;L evans blue solution to the cell culture insert.</li>
		<li>Add 600 &#181;L PBS/B27 to the lower compartment and incubate for 60 min at 37 &#176;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 &#181;L 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 &#181;s).</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 &#181;L 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 &#181;L 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 &#181;L RPMI/B27 to the lower compartment of the cell culture inserts and 500 &#181;L to the PBMC of the in vitro control and incubate 6 h at 37 &#176;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 &#181;L PBS without touching the membrane. Discard the cell culture insert.</li>
		<li>Add 20 &#181;L flow count fluorospheres (approximately 1,000 beads/&#181;L) 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 &#181;L flow cytometry buffer (PBS/1% BSA/2 mM EDTA) per sample. For the results presented below 1 &#181;L CD4-FITC, 1 &#181;LCD3-PerCP/Cy5.5, 1 &#181;L CD56-PC7, 1 &#181;L CD8-A700, and 1 &#181;L CD16-A750 were used per sample.</li>
		<li>Re-suspend PBMC in 100 &#181;L of the antibody solution and incubate for 30 min at 4 &#176;C.</li>
		<li>Add 250 &#181;L 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 &#34;cytometer on&#34; button. (3) Load the respective acquisition protocol by pressing &#34;open protocol&#34; button. (4) Choose the required protocol and select &#34;open&#34;. (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 &#34;duplicate&#34;. (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="Equation" src="/files/ftp_upload/55390/55390eq1.jpg" /></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 cell population. Furthermore, treatment of the HBMEC monolayer with IFN-&#947; and TNF-&#945; 24 hours prior to the assay can be used to mimic an inflamed blood-brain barrier in order to study the effect of inflammatory CNS disorders21,25. Similarly, treatment of HBMEC or lymphocytes with other substances allows investigating their effects on lymphocyte extravasation (e.g. their effect on adhesion molecules)30,31. The involvement of certain adhesion molecules can be studied using blocking antibodies for integrins or their ligands32. Furthermore, the experimental setup presented here allows analysis of chemotactic effects from chemokines or supernatants derived from astrocytes or other cells33,34. Replacement of HBMEC with primary human brain derived epithelial cells widens the spectrum of this experimental setup for investigation of the blood-cerebrospinal fluid barrier15. HBMEC should not be replaced with immortalized endothelial cells or cells derived from other organs to maintain CNS-specificity of the model. However, brain-derived endothelial cells from other species might be used to analyze transmigration in the respective animals35. In addition, retinoic acid or hydrocortisone have been described to increase barrier functions and might thus be employed36,37. In case of limited cell numbers the amount of cells subjected to the assay might be reduced, because our model provides linear recovery rates between 2 x 105 and 1 x 106 PBMC (data not shown). To analyze rare cell populations following transmigration it might be necessary to pool cells from several wells in order to obtain sufficient cells required for flow cytometry. Finally, our experimental setup could also be used to study the drug delivery into the CNS38. A pore size of 3 &#181;m is typically used to allow lymphocyte transmigration, whereas a pore size of 0.4 &#181;m prevents lymphocyte transmigration, but allows to study drug delivery39,40,41,42.
Despite the various applications of the experimental setup described here, several points have to be kept in mind to ensure meaningful results. The integrity of the porous membrane as well as the HBMEC monolayer is critical. Thus, it is mandatory to avoid physical damage of the membrane, for example with pipet tips. Addition of fluids or cell suspension to the upper part of the cell culture insert should be performed without direct contact. Droplets might be wiped off at the border of the cell culture insert. Horizontal placement of the pipet tip ensures protection of the membrane, when the abluminal membrane is rinsed at the end of the migration assay. The quality of the HBMEC monolayer can be analyzed by microscopic evaluation of confluence as well as evans blue permeation as demonstrated for the assessment of blood-brain barrier integrity in rodent experiments43. Centrifugation of HBMEC at low g forces and usage of early passages (i.e. 1 - 15) ensures quality and uniformity of the monolayer. To prevent dissolution from the porous membrane, it is important to adhere to the described order of aspiration and addition of medium, when working with the HBMEC monolayer.
Although adherence to the protocol described here ensures meaningful and reproducible results, this technique has some limitations. First of all, in vivo the blood-brain barrier is formed by a number of cells, which interact in various ways and strengthen the barrier function by affecting the formation of tight junctions1. Therefore, although this blood-brain barrier model is a good approximation of the in vivo situation, important aspects are missing. In addition, lymphocyte extravasation into the CNS across the blood-brain barrier is a multistep process. While each single step can be investigated separately, the technique presented here does not provide information on the whole process of extravasation under the influence of shear forces2,44,45,46. Finally, analysis of migrated cells from PBMC might be challenging depending on the frequency of the cells of interest, because usually only single-digit frequencies of cells transmigrate. Therefore, separation of the populations of interest prior to the assay or pooling of cells migrated across multiple cell culture inserts might be necessary. Other models for the analysis of leukocyte migration into the CNS cover some of the aspects missing in our model. Co-culture with astrocytes, pericytes, and/or neurons are used to mimic the complexity of the blood-brain barrier better47. Models including shear forces such as DIV-BBB reflect more the physiological conditions, thus, enabling a more sophisticated analysis of lymphocyte diapedesis48.
In summary, we present an easily accessible technique suitable for the investigation of qualitative and quantitative lymphocyte diapedesis across the human blood-brain barrier.

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>PBS</td>
			<td>Gibco</td>
			<td>14190-094</td>
			<td>without CaCl2 or MgCl2</td>
		</tr>
		<tr>
			<td>Fibronectin 1mg/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, 6.5mm diameter, 3.0&#181;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-&#945;</td>
			<td>Peprotech</td>
			<td>300-01A</td>
			<td></td>
		</tr>
		<tr>
			<td>IFN-&#947;</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

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). Furthermore, HBMEC grew in monolayers exhibiting transendothelial electrical resistance (Figure 1B middle) as well as reduced permeation for evans blue (Figure 1B, right). HBMEC monolayers were used to study the transmigration of NK cells including CD56brightCD16dim/- and CD56dimCD16+ NK-cell subsets and T cells including CD4+ and CD8+ T cells as two examples (Figure 1D+E, respectively). The percentage of migrated cells was calculated based on cell counts obtained by flow cytometry and normalized using flow count fluorospheres as an internal control (Figure 1C). The HBMEC monolayer was stimulated with IFN-&#947; and TNF-&#945; 24h prior the assay to mimic inflammatory conditions. Cytokine stimulation resulted in increased migration of all analyzed lymphocyte populations. This might be due to an increased expression of adhesion molecules including ICAM-1 (data not shown). CD56brightCD16dim/- NK cells exhibited a higher migratory capacity when compared to their CD56dimCD16+ NK across both unstimulated (10.88% vs. 0.86%) and IFN-&#947;&#947;/TNF-&#945; stimulated (18.22% vs. 2.94%) HBMEC (Figure 1D). However, the relative increase of transmigration as a result of inflammation was higher for the CD56dimCD16+ NK-cell subset (+342% vs. +167% for CD56brightCD16dim/-). These results mimic the in vivo observations that CD56brightCD16dim/- NK cells are enriched in the intrathecal compartment. Thus, the blood-brain barrier model seems to be suitable to analyze basic principles of immune-cell diapedesis of rare lymphocyte populations into the CNS21. Finally, the transmigratory capacity of CD4 and CD8 T-cell subsets is shown (Figure 1E).
<img alt="Figure 1" src="/files/ftp_upload/55390/55390fig1.jpg" /> 
Figure 1: Differential Migration of NK-cell Subsets across Uninflamed and Inflamed HBMEC Monolayers. A. A picture of transwell inserts (left) and illustration of the experimental setup (right). B. Validation of HBMEC barrier functions. Left: immunohistochemical staining of the tight junction molecule ZO-1 using rabbit anti-human ZO-1 (abcam, 1:200) and goat anti-rabbit IgG-Cy3 (1:300) on HBMEC cultivated for 3 days. Center: transendothelial electrical resistance (TEER) of HBMEC between day 2 and day 4 of cultivation. Right: standard curve for evans blue permeation for HBMEC with (&#34;inflamed&#34;, red) or without (&#34;uninflamed&#34;, black) stimulation with 500 U/mL IFN-&#947; and TNF-&#945; for 24 hours. Transmigration assays are performed 72 - 96 hours after seeding of the HBMEC (black arrow). C-E. PBMC derived from 16 healthy individuals were subjected to migration assays as described in the protocol section. 24 h prior to the assay, half of the cell culture inserts were stimulated with 500 IU/mL IFN-&#947; and TNF-&#945;. Transmigrated cells were harvested and analyzed by flow cytometry. C. Representative results for PBMC derived from the in vitro control well (top) and after migration across uninflamed HBMEC (bottom). NK cells were gated from total lymphocytes as CD3-CD56+ cells and further distinguished into CD56brightCD16dim/- (&#34;CD56bright&#34;) and CD56dimCD16+ (&#34;CD56dim&#34;) NK-cell subsets. Flow count fluorospheres (&#34;beads&#34;) were gated based on FSC/SSC characteristics and their number was determined in a FL3 versus time plot. Exemplary calculations to determine the percentage of migrated CD56bright and CD56dim NK cells are shown on the right. D. Percentage of migrated NK cells as well as CD56bright and CD56dim NK-cell subsets, and E. percentage of migrated T cells including CD4+ and CD8+ T-cell subsets following transmigration across uninflamed (black) or inflamed (red) HBMEC depicted as mean &#177; SEM. P-values were calculated by paired student t-test; **p&#60;0.01, ***p&#60;0.001. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55390/55390fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier at JoVE.com

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.

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 ...

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Nanyang Technological University

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JoVE Journal

Immunology and Infection

20.3K Views.  University Hospital Münster. Here, we describe a human blood-brain barrier model enabling to investigate lymphocyte transmigration into the central nervous system in vitro. 

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

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular matrix proteins. The precise spatiotemporal activation of integrins from a low affinity state to a high affinity state at the cell leading edge is important for T lymphocyte migration 1. Likewise, retraction of the cell trailing edge, or uropod, is a necessary step in maintaining persistent integrin-dependent T lymphocyte motility 2. Many therapeutic approaches to autoimmune or inflammatory diseases target integrins as a means to inhibit the excessive recruitment and migration of leukocytes 3. To study the molecular events that regulate human T lymphocyte migration, we have utilized an in vitro system to analyze cell migration on a two-dimensional substrate that mimics the environment that a T lymphocyte encounters during recruitment from the vasculature. T lymphocytes are first isolated from human donors and are then stimulated and cultured for seven to ten days. During the assay, T lymphocytes are allowed to adhere and migrate on a substrate coated with intercellular adhesion molecule-1 (ICAM-1), a ligand for integrin LFA-1, and stromal cell-derived factor-1 (SDF-1). Our data show that T lymphocytes exhibit a migratory velocity of ~15 &mu;m/min. T lymphocyte migration can be inhibited by integrin blockade 1 or by inhibitors of the cellular actomyosin machinery that regulates cell migration 2.

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

T lymphocyte migration occurs during homing to lymphoid organs, exit from the vasculature, and entering into peripheral tissues. Here, we describe a protocol that can be used to analyze T lymphocyte migration in vitro.

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular ...

Finger-stick Blood Sampling Methodology for the Determination of Exercise-induced Lymphocyte Apoptosis

Exercise is a physiological stimulus capable of inducing apoptosis in immune cells. To date, various limitations have been identified with the measurement of this phenomenon, particularly relating to the amount of time required to isolate and treat a blood sample prior to the assessment of cell death. Because of this, it is difficult to determine whether reported increases in immune cell apoptosis can be contributed to the actual effect of exercise on the system, or are a reflection of the time and processing necessary to eventually obtain this measurement. In this article we demonstrate a rapid and minimally invasive procedure for the analysis of exercise-induced lymphocyte apoptosis. Unlike other techniques, whole blood is added to an antibody panel immediately upon obtaining a sample. Following the incubation period, red blood cells are lysed and samples are ready to be analyzed. The use of a finger-stick sampling procedure reduces the volume of blood required, and minimizes the discomfort to subjects.

Exercise is capable of inducing apoptosis in immune cells. There are various measurement limitations, particularly relating to the amount of time required to isolate and treat a blood sample prior to the assessment. Demonstrated is a rapid and minimally invasive procedure for the analysis of exercise-induced lymphocyte apoptosis.

Exercise is a physiological stimulus capable of inducing apoptosis in immune cells. To date, various limitations have been identified with the measurement ...

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response following an infection. Despite efforts to identify unique surface marker on Tregs, the only unique feature is their ability to suppress the proliferation and function of effector T cells. While it is clear that only in vitro assays can be used in assessing human Treg function, this becomes problematic when assessing the results from cross-sectional studies where healthy cells and cells isolated from subjects with autoimmune diseases (like Type 1 Diabetes-T1D) need to be compared. There is a great variability among laboratories in the number and type of responder T cells, nature and strength of stimulation, Treg:responder ratios and the number and type of antigen-presenting cells (APC) used in human in vitro suppression assays. This variability makes comparison between studies measuring Treg function difficult. The Treg field needs a standardized suppression assay that will work well with both healthy subjects and those with autoimmune diseases. We have developed an in vitro suppression assay that shows very little intra-assay variability in the stimulation of T cells isolated from healthy volunteers compared to subjects with underlying autoimmune destruction of pancreatic &beta;-cells. The main goal of this piece is to describe an in vitro human suppression assay that allows comparison between different subject groups. Additionally, this assay has the potential to delineate a small loss in nTreg function and anticipate further loss in the future, thus identifying subjects who could benefit from preventive immunomodulatory therapy1. Below, we provide thorough description of the steps involved in this procedure. We hope to contribute to the standardization of the in vitro suppression assay used to measure Treg function. In addition, we offer this assay as a tool to recognize an early state of immune imbalance and a potential functional biomarker for T1D.

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Tregs are potent suppressors of the immune system. There is a lack of unique surface markers to define them, hence, definitions of Tregs are primarily functional. Here we describe an optimized in vitro assay capable of identifying immune imbalance in subjects at risk to develop T1D.

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response ...

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the phenotypic and functional differences between murine and human monocyte-derived macrophages. Therefore, we here describe an in vitro model to generate and study primary human macrophages. Briefly, after density gradient centrifugation of peripheral blood drawn from a forearm vein, monocytes are isolated from peripheral blood mononuclear cells using negative magnetic bead isolation. These monocytes are then cultured for six days under specific conditions to induce different types of macrophage differentiation or polarization. The model is easy to use and circumvents the problems caused by species-specific differences between mouse and man. Furthermore, it is closer to the in vivo conditions than the use of immortalized cell lines. In conclusion, the model described here is suitable to study macrophage biology, identify disease mechanisms and novel therapeutic targets. Even though not fully replacing experiments with animals or human tissues obtained post mortem, the model described here allows identification and validation of disease mechanisms and therapeutic targets that may be highly relevant to various human diseases.

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Monocyte-derived macrophages are important cells of the innate immune system. Here, we describe an easy to use in vitro model to generate these cells. Using gradient centrifugation, negative bead isolation and specific cell culture conditions, monocyte-derived macrophages can be generated for phenotypic and functional studies.

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the ...

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) infections present a broad spectrum of disease severity, ranging from mild infections to life-threatening bronchiolitis. An important part of the pathogenesis of severe disease is an enhanced immune response leading to immunopathology.	Here, we describe a protocol used to investigate the immune response of human immune cells to an HRSV infection. First, we describe methods used for culturing, purification and quantification of HRSV. Subsequently, we describe a human in vitro model in which peripheral blood mononuclear cells (PBMCs) are stimulated with live HRSV. This model system can be used to study multiple parameters that may contribute to disease severity, including the innate and adaptive immune response. These responses can be measured at the transcriptional and translational level. Moreover, viral infection of cells can easily be measured using flow cytometry. Taken together, stimulation of PBMC with live HRSV provides a fast and reproducible model system to examine mechanisms involved in HRSV-induced disease.

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) can cause severe bronchiolitis in young infants. Part of the pathogenesis of severe HRSV disease is caused by the host immune response. Stimulation of primary human immune cells with HRSV provides a fast and reproducible model system to study activation of inflammatory pathways and infection.

Human respiratory syncytial  virus (HRSV) infections present a broad spectrum of disease severity, ranging  from mild infections to life-threatening ...

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 ...

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 ...

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the skin is relatively easy to access, it provides an ideal platform to study peripheral immune regulatory mechanisms. Immune resident cells in healthy skin conduct immunosurveillance, but also play an important role in the development of inflammatory skin disorders, such as psoriasis. Despite emerging insights, our understanding of the biology underlying various inflammatory skin diseases is still limited. There is a need for good quality (single) cell populations isolated from biopsied skin samples. So far, isolation procedures have been seriously hampered by a lack of obtaining a sufficient number of viable cells. Isolation and subsequent analysis have also been affected by the loss of immune cell lineage markers, due to the mechanical and chemical stress caused by the current dissociation procedures to obtain single cell suspension. Here, we describe a modified method to isolate T cells from both healthy and involved psoriatic human skin by combining mechanical skin dissociation using an automated tissue dissociator and collagenase treatment. This methodology preserves expression of most immune lineage markers such as CD4, CD8, Foxp3 and CD11c upon the preparation of single cell suspensions. Examples of successful CD4+ T cell isolation and subsequent phenotypic and functional analysis are shown.

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

We describe a protocol to efficiently isolate skin resident T cells from human skin biopsies. This protocol yields sufficient numbers of viable human skin resident lymphocytes for flow cytometric analysis and ex vivo culture.

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the ...

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.

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.

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 ...

Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining the role of the gene of interest in the development, differentiation, and function of NK cells. Recent advances related to chimeric antigen receptors (CARs) in cancer immunotherapy accentuate the need for an efficient method to deliver exogenous genes to effector lymphocytes. The efficiencies of lentiviral-mediated gene transductions into primary human or mouse NK cells remain significantly low, which is a major limiting factor. Recent advances using cationic polymers, such as polybrene, show an improved gene transduction efficiency in T cells. However, these products failed to improve the transduction efficiencies of NK cells. This work shows that dextran, a branched glucan polysaccharide, significantly improves the transduction efficiency of human and mouse primary NK cells. This highly reproducible transduction methodology provides a competent tool for transducing human primary NK cells, which can vastly improve clinical gene delivery applications and thus NK cell-based cancer immunotherapy.

The goal of this study was to formulate technologies that allow for successful gene transduction in primary natural killer (NK) cells. The dextran-mediated lentiviral transduction of human or mouse primary NK cells results in higher gene expression efficiencies. This method of gene transduction will vastly improve NK cell genetic manipulation.

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining ...