Name: studying lymphocyte transmigration across a microvascular endothelial cell monolayer
Uploaded: 2025-03-31T15:00:45.000+00:00
Duration: 1 min 24 s
Description: Source: Schulte-Mecklenbeck, A., et al., Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier. J. Vis. Exp. ...

studying lymphocyte transmigration across a microvascular endothelial cell monolayer

Studying Lymphocyte Transmigration Across a Microvascular Endothelial Cell Monolayer

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. Resuspend the Peripheral Blood Mononuclear Cell, or PBMC sample to a 5 times 10 to the 6 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 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 1 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 4 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.

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1. 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 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 endothelial cell medium-basal (ECM-b) medium to the lower compartment of the cell culture inserts. Incubate for 3 - 4 days at 37 &#176;C / 5% CO2&#160;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>
	</ol>
	</li>
</ol>
2. Migration Assay
<ol>
	<li>Preparation of peripheral blood mononuclear cells (PBMC).
	<ol>
		<li>Add 10 mL Roswell Park Memorial Institute (RPMI) medium 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&#160;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&#160;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&#160;in vitro&#160;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 phosphate-buffered saline (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&#160;in vitro&#160;control and mix well. Transfer 1 mL of resulting PBMC suspension to flow cytometry tubes.</li>
	</ol>
	</li>
</ol>
3. 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% bovine serum albumin [BSA]/2 mM ethylenediaminetetraacetic acid [EDTA]) per sample. For the results presented below 1 &#181;L CD4-FITC (cluster of differentiation 4-fluorescein isothiocyanate), 1 &#181;L CD3-PerCP/Cy5.5 (peridinin chlorophyll protein/cyanine 5. 5), 1 &#181;L CD56-PC7 (phycoerythrin cyanin 7), 1 &#181;L CD8-A700 (Alexa fluor 700), and 1 &#181;L CD16-A750 (Alexa fluor 750), 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>
</ol>

<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>HBMEC</td>
			<td>ScienCell</td>
			<td>1000</td>
			<td></td>
		</tr>
		<tr>
			<td>HBMEC</td>
			<td>Pelobiotech</td>
			<td>PB-H-6023</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>B27</td>
			<td>Gibco</td>
			<td>17504-044</td>
			<td>50x concentrated</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>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>

Source: Schulte-Mecklenbeck, A., et al., <a href="https://app.jove.com/v/55390">Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier.</a> J. Vis. Exp. (2017)
This video demonstrates an experimental investigation of lymphocyte transmigration across a blood-brain barrier (BBB) endothelium model. A monolayer of human brain microvascular endothelial cells (HBMECs) is generated in vitro, and peripheral blood mononuclear cells (PBMCs) containing lymphocytes are added to the monolayer. The cells are then incubated to allow lymphocyte transmigration, and the migrated cells are quantified using flow cytometry.

<img alt="Figure 1" src="/files/ftp_upload/23131/23131_Figure1.jpg" /> 
Figure 1: Differential Migration of NK-cell Subsets across Uninflamed and Inflamed HBMEC Monolayers. A.&#160;A picture of transwell inserts (left) and illustration of the experimental setup (right).&#160;B.&#160;Validation of HBMEC barrier functions. Left: immunohistochemical staining of the tight junction molecule ZO-1 using rabbit anti-human zonula occludens-1 (ZO-1, abcam, 1:2...

Source: Schulte-Mecklenbeck, A., et al., Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier. J. Vis. Exp. ...

Take a microplate with transwell inserts coated with ECM proteins to promote cell adhesion.
Add Human brain microvascular endothelial cells, or HBMECs , into the inserts and a growth medium to the lower compartments.
Incubate, allowing the cells to adhere and grow into a monolayer. These cells express tight junctions, mimicking the blood-brain barrier endothelium that limits the passage of cells and molecules.
Remove the medium. Add peripheral blood mononuclear cells, or PBMCs, to the inserts and a culture medium to the lower compartments.
Incubate, allowing PBMC lymphocytes to interact with the endothelial cells and then pass between them, a process called transmigration.
Rinse the insert bottom, collecting the adhered transmigrated cells into the lower compartments.
Introduce fluorescent microbeads as a reference for cell quantification.
Transfer the suspension into a tube, centrifuge, discard the supernatant, and resuspend in a fluorophore-conjugated antibody cocktail to label the lymphocytes.
The transmigrated lymphocytes are ready for quantification via flow cytometry.

9 Views. Studying Lymphocyte Transmigration Across a Microvascular Endothelial Cell Monolayer

Video: Studying Lymphocyte Transmigration Across a Microvascular Endothelial Cell Monolayer - Experiment

Isolation and Enrichment of Liver Progenitor Subsets Identified by a Novel Surface Marker Combination

During chronic liver injuries, progenitor cells expand in a process called ductular reaction, which also entails the appearance of inflammatory cellular infiltrate and epithelial cell activation. The progenitor cell population during such inflammatory reactions has mostly been investigated using single surface markers, either by histological analysis or by flow cytometry-based techniques. However, novel surface markers identified various functionally distinct subsets within the liver progenitor/stem cell compartment. The method presented here describes the isolation and detailed flow cytometry analysis of progenitor subsets using novel surface marker combinations. Moreover, it demonstrates how the various progenitor cell subsets can be isolated with high purity using automated magnetic and FACS sorting-based methods. Importantly, novel and simplified enzymatic dissociation of the liver allows for the isolation of these rare cell populations with a high viability that is superior in comparison to other existing methods. This is especially relevant for further studying progenitor cells in vitro or for isolating high-quality RNA to analyze the gene expression profile.

Liver injuries are accompanied by progenitor cell expansion that represents a heterogeneous cell population. Novel classification of this cellular compartment allows for the distinguishing of multiple subsets. The method described here illustrates the flow cytometry analysis and high purity isolation of various subsets that can be used for further assays.

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Quantification of Monocyte Transmigration and Foam Cell Formation from Individuals with Chronic Inflammatory Conditions

Coronary artery disease (CAD) is a leading cause of morbidity and mortality worldwide. Atherosclerosis, a leading cause of CAD, is initiated by the transmigration of innate immune monocytes to inflammatory sites of deposited lipid called fatty streaks, which are present in arterial walls of medium to large arteries. The key pathogenic feature of lesions at this early stage of atherosclerosis is the maturation of monocytes which migrate into arteries to form foam cells or lipid-laden macrophages. Considerable evidence supports the hypothesis that risk of atherosclerosis is increased by chronic inflammatory conditions accompanying diseases such as rheumatoid arthritis and HIV, as well as general ageing, and that this risk is predicted by monocyte activation. While mouse models provide a good platform to investigate the role of monocytes in atherogenesis in vivo, they require genetic alteration of natural cholesterol metabolism and drastic alteration of normal mouse diets, and have limited suitability for the study of atherogenic influences of human comorbid diseases. This motivated us to develop a human in vitro model to measure the atherogenic potential of monocytes isolated from individuals with defined disease states. Currently, human in vitro models are limiting in that they evaluate monocyte transmigration and foam cell formation in isolation. Here we describe a protocol in which monocytes isolated from patient blood transmigrate across human endothelial cells into a type 1 collagen matrix, and their propensity to mature into foam cells in the presence or absence of exogenous lipid is measured. The protocol has been validated for the use of human monocytes purified from individuals with HIV infection and elderly HIV uninfected individuals. This model is versatile and allows monocyte transmigration and foam cell formation to be evaluated using either microscopy or flow cytometry as well as allowing the assessment of atherogenic factors present in serum or plasma.

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Herein, we present an efficient method that can be executed with basic laboratory skills and materials to assess lymphocyte chemokinetic movement in an ex vivo transmigration system. Group 2 innate lymphoid cells (ILC2) and CD4+ T helper cells were isolated from spleens and lungs of chicken egg ovalbumin (OVA)-challenged BALB/c mice. We confirmed the expression of CCR4 on both CD4+ T cells and ILC2, comparatively. CCL17 and CCL22 are the known ligands for CCR4; therefore, using this ex vivo transmigration method we examined CCL17- and CCL22-induced movement of CCR4+ lymphocytes. To establish chemokine gradients, CCL17 and CCL22 were placed in the bottom chamber of the transmigration system. Isolated lymphocytes were then added to top chambers and over a 48 h period the lymphocytes actively migrated through 3 &#181;m pores towards the chemokine in the bottom chamber. This is an effective system for determining the chemokinetics of lymphocytes, but, understandably, does not mimic the complexities found in the in vivo organ microenvironments. This is one limitation of the method that can be overcome by the addition of in situ imaging of the organ and lymphocytes under study. In contrast, the advantage of this method is that is can be performed by an entry-level technician at a much more cost-effective rate than live imaging. As therapeutic compounds become available to enhance migration, as in the case of tumor infiltrating cytotoxic immune cells, or to inhibit migration, perhaps in the case of autoimmune diseases where immunopathology is of concern, this method can be used as a screening tool. In general, the method is effective if the chemokine of interest is consistently generating chemokinetics at a statistically higher level than the media control. In such cases, the degree of inhibition/enhancement by a given compound can be determined as well.

In this protocol, lymphocytes are placed in the top chamber of a transmigration system, separated from the bottom chamber by a porous membrane. Chemokine is added to the bottom chamber, which induces active migration along a chemokine gradient. After 48 h, lymphocytes are counted in both chambers to quantitate transmigration.

Herein, we present an efficient method that can be executed with basic laboratory skills and materials to assess lymphocyte chemokinetic movement in an ex ...

Studying Lymphocyte Transmigration Across a Microvascular Endothelial Cell Monolayer

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Studying Lymphocyte Transmigration Across a Microvascular Endothelial Cell Monolayer