The authors thank Dr. P.L. Hordijk for critically reading the manuscript. AED is supported by a Landsteiner Foundation for Blood Transfusion Research (LSBR) fellowship (grant #1028). JK is supported by the Dutch Heart Foundation (2005T3901). JDvB is a NHS Dekker fellow (grant #2005T039).

Neutrophils were isolated from healthy volunteers&#160;who have signed an informed consent. Research has been performed in compliance with the institutional and national guidelines for human welfare.
1. Plating and Maintenance of Human Umbilical Vein Endothelial Cells
<ol>
	<li>Culture Human Umbilical Vein Endothelial Cells (HUVECs)&#160;according to manufacturer&#8217;s instructions. Grow HUVECs on fibronectin (FN)-coated dishes (10 &#956;g/ml, dissolved in demineralized water) using media (Endothelial Basal Medium (EBM-2) medium supplemented with Endothelial Growth Medium (EGM-2) singlequots). Use cell culture between 4-8 passages for experiments.</li>
	<li>Day 1: Coat flow-chambers with 100 &#956;l&#160;of fibronectin (10 &#956;g/ml&#160;in PBS) for at least 2 hr at 37 &#176;C and 5% CO2.</li>
	<li>Day 2: When cells reach 80-90% confluence, trypsinize the cells after careful washing with RT phosphate buffered saline (PBS) pH 7.4, centrifuge at 800 x g and resuspend at 800,000 cells/ml&#160;using media. Plate 80,000 cells in every single channel of the FN-coated flow-chambers and gently pipette the cell suspension up and down. Culture O/N&#160;in an incubator at 37 &#176;C and 5% CO2.</li>
	<li>Day 3: Refresh the media in the flow-chamber slide by gently tilting the slide at a 45&#176; angle. (see Figure 1A). 
	NOTE: It is recommended to only remove the media in the reservoirs and not in the channel itself. Removal of the media in the channels may result in endothelial cell loss and death due to the dragging force of the media&#160;caused by pipetting.</li>
	<li>Check by phase contrast microscopy if the endothelial cells have formed a monolayer (i.e., 100% confluent). If the cells are not 100% confluent, change the media two times a day until they reach 100% confluence.</li>
	<li>Once the cells have reached confluency, stimulate the cells with media (see step 1.1) containing inflammatory mediator (TNF-&#945; (10 ng/ml)). Stimulating HUVECs O/N (i.e., 12 hr) with TNF-&#945; results in an inflammatory phenotype of the endothelium, i.e., up-regulation of cell adhesion molecules such as ICAM-1 and VCAM-1 13.</li>
</ol>
2. Polymorphonuclear Leukocyte Isolation Using Percoll Gradients
<ol>
	<li>Prepare N-2-hydroxyethylpiperazine-N&#39;-2-ethanesulfonic acid (HEPES)-buffer (hereafter called: flow-buffer) before isolating polymorphonuclear leukocytes (PMN). Flow-buffer is used to wash isolated PMNs and as buffer in the flow assay. To prepare flow-buffer: dilute 7.72 g NaCl (132 mM), 4.76 g HEPES (20 mM), 0.45 g KCl (6 mM), 0.25 g MgSO4&#8226;7H2O (1 mM), K2HPO4&#8226;3H2O (1.2 mM) in 1 L of demineralized water and adjust to pH 7.4 (this stock can be kept at 4 &#176;C for several weeks).</li>
	<li>Add fresh 100 &#956;l&#160;1 M CaCl2&#160;(1 mM), 2.5 ml&#160;human albumin from a 200 g/L stock concentration (0.5% v/v) and 0.1 g glucose to 100 ml&#160;(0.1% w/v) of flow-buffer. Next, filter the flow-buffer using a 0.45 &#956;m filter. NOTE: This flow-buffer needs to be prepared fresh for every experiment.</li>
	<li>Prior to isolation of PMN, prepare 10% trisodium citrate (TNC) solution in PBS, pH 7.4.</li>
	<li>Collect 20 ml&#160;of whole blood in sodium heparine vacuettes from a healthy volunteer. Dilute whole blood 1:1 with 10% PBS/TNC in 50 ml&#160;tubes and pipette 20 ml&#160;diluted blood carefully onto 12.5 ml&#160;of Percoll (a 23% (w/w) colloidal solution in water with&#160;a density of 1.130 g/ml) in a new 50 ml&#160;tube. Carefully place the tubes in the centrifuge and spin for 20 min at 800 x g with low acceleration and no break set at RT. 
	NOTE: When adding the diluted blood to the Percoll, tilt the Percoll-containing tube in a 45&#176; angle and gently pipette the diluted blood in the tube with the slowest pipette boy setting.</li>
	<li>Remove all liquid and subsequently fill the tube with ice-cold erythrocyte lysis-buffer (4.15 g NH4Cl [0.155 M], 0.5 g KHCO3 [0.01 M] and 18.5 mg EDTA (triplex III) [0.1 mM] to 500 ml&#160;of ice-cold H2O) to lyse erythrocytes. Leave tube on ice, occasionally inverting the tube, until the suspension turns dark red, followed by centrifugation 500 x g for 5 min at 4&#176;C with breaks enabled. 
	NOTE: The pellet fraction contains the PMNs (neutrophils, eosinophils, basophils) together with the erythrocytes.</li>
	<li>Remove supernatant and wash pellet twice in ice-cold lysis-buffer at 500 x g for 5 min at 4 &#176;C.</li>
	<li>Resuspend the pellet with flow-buffer at RT and determine PMNs concentration using either a hemocytometer or automated cell counter. Suspend the PMNs in flow-buffer at 1 x 106 cells/ml&#160;and keep at RT.</li>
</ol>
3. Labeling of Endothelial Junctional VE-cadherin and PECAM-1
<ol>
	<li>Add the PECAM Alexa Fluor 647 antibody (clone WM59) at a 1:100 dilution and the calcium-independent VE-cadherin-FITC antibody (clone 55-7H1) at a 1:50 dilution to HUVEC culture medium (see step 1.1), and incubate for 30 min prior to starting the flow experiment to visualize junctional dynamics of endothelial cells during neutrophil transmigration under physiological flow conditions. 
	NOTE: Before adding the directly-labeled antibodies to the flow-chambers, ensure that the volume in each channel does not exceed 100 &#956;l. This helps keep&#160;the antibody expenses as low as possible.</li>
</ol>
4. PMN TEM Assay Under Flow
<ol>
	<li>Place the PMNs into a water bath for 15 min at 37 &#176;C prior to injecting them into the flow-system.</li>
	<li>Connect the tubing to an empty flow-chamber and fill with warm (e.g., 37 &#176;C) flow-buffer (see step 4.3) to prevent formation of air-bubbles when setting up the flow system.</li>
	<li>Connect one side of an empty flow-chamber to the syringe pump flow system by using silicone tubing&#160;containing a 20 ml&#160;syringe, and put the flow-chamber into the microscope stage.</li>
	<li>Connect the other side of an empty flow-chamber to the reservoir flask filled with 37 &#176;C flow-buffer (Figure 1B) and start the syringe pump in order to fill all the tubing with flow-buffer. The pump will pull the flow-buffer from the reservoir through the flow-chamber&#160;into the syringe. NOTE: This tubing also contains an in-line Luer injection port, which allows PMNs to be injected with a needle into a running experiment without stopping the flow and creating air bubbles.</li>
	<li>Replace the empty flow-chamber with the flow-chamber containing TNF-&#945;-treated HUVECs, connect the flow-buffer-containing tubing and place it into the microscope stage (Figure 1C). Pinch off the tubes before disconnecting and reconnecting them to the chambers containing the HUVECs, as not pinching off may result in the formation of air bubbles inside the tubing and/or flow-chambers.</li>
	<li>Adjust the flow speed to 1 dyn/cm2, in accordance with physiological flow speed in post capillary venules (1-5 dyn/cm2).</li>
	<li>Record Differential Interference Contrast (DIC), FITC (488 nm) and Alexa Fluor 647 (647 nm) simultaneously using a confocal laser scanning microscope.</li>
	<li>Inject the PMNs (step 4.1) slowly in the flow-system via the in-line Luer injection port (Figure 1D) using 1 ml&#160;syringes.</li>
	<li>After a few minutes, leukocytes appear, adhere, and transmigrate. Stop the experiment at any desired moment by disconnecting the tubing from the flow-chamber and pipetting fixative (3.7% formaldehyde in PBS) into the flow-chamber. Allow fixation for 10 min, followed by washing with PBS. Data is analyzed using imaging software (see materials and equipment&#160;table). 
	NOTE: Perform the he experiment using a confocal laser scanning microscope equipped with a climate chamber&#160;with a constant temperature at 37 &#176;C, 5% CO2,&#160;and a 63X&#160;oil-objective.</li>
</ol>

<ol>
	<li>Ross, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atherosclerosis--an+inflammatory+disease.">Atherosclerosis--an inflammatory disease.</a> N. Engl. J. Med. 340, 115-126 (1999).</li><li>Luster, A. D., Alon, R., von Andrian, U. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+cell+migration+in+inflammation:+present+and+future+therapeutic+targets.">Immune cell migration in inflammation: present and future therapeutic targets.</a> Nat. Immunol. 6, 1182-1190 (2005).</li><li>Szekanecz, Z., Koch, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+involvement+in+rheumatic+diseases:+'+vascular+rheumatology.">Vascular involvement in rheumatic diseases: ' vascular rheumatology.</a> Arthritis Res. Ther. 10, 224 (2008).</li><li>Butcher, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leukocyte-endothelial+cell+recognition:+three+(or+more)+steps+to+specificity+and+diversity.">Leukocyte-endothelial cell recognition: three (or more) steps to specificity and diversity.</a> Cell. 67, 1033-1036 (1991).</li><li>Springer, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Traffic+signals+for+lymphocyte+recirculation+and+leukocyte+emigration:+the+multistep+paradigm.">Traffic signals for lymphocyte recirculation and leukocyte emigration: the multistep paradigm.</a> Cell. 76, 301-314 (1994).</li><li>Ley, K., Laudanna, C., Cybulsky, M. I., Nourshargh, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Getting+to+the+site+of+inflammation:+the+leukocyte+adhesion+cascade+updated.">Getting to the site of inflammation: the leukocyte adhesion cascade updated.</a> Nat. Rev. Immunol. 7, 678-689 (2007).</li><li>Cinamon, G., Shinder, V., Shamri, R., Alon, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemoattractant+signals+and+beta+2+integrin+occupancy+at+apical+endothelial+contacts+combine+with+shear+stress+signals+to+promote+transendothelial+neutrophil+migration.">Chemoattractant signals and beta 2 integrin occupancy at apical endothelial contacts combine with shear stress signals to promote transendothelial neutrophil migration.</a> J. Immunol. 173, 7282-7291 (2004).</li><li>Shulman, Z., Cohen, S. J., Roediger, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transendothelial+migration+of+lymphocytes+mediated+by+intraendothelial+vesicle+stores+rather+than+by+extracellular+chemokine+depots.">Transendothelial migration of lymphocytes mediated by intraendothelial vesicle stores rather than by extracellular chemokine depots.</a> Nat. Immunol. 13, 67-76 (2012).</li><li>Carman, C. V., Springer, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trans-cellular+migration:+cell-cell+contacts+get+intimate.">Trans-cellular migration: cell-cell contacts get intimate.</a> Curr. Opin. Cell Biol. 20, 533-540 (2008).</li><li>Carman, C. V., Springer, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+transmigratory+cup+in+leukocyte+diapedesis+both+through+individual+vascular+endothelial+cells+and+between+them.">A transmigratory cup in leukocyte diapedesis both through individual vascular endothelial cells and between them.</a> J. Cell Biol. 167, 377-388 (2004).</li><li>Millan, J., Hewlett, L., Glyn, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphocyte+transcellular+migration+occurs+through+recruitment+of+endothelial+ICAM-1+to+caveola-+and+F-actin-rich+domains.">Lymphocyte transcellular migration occurs through recruitment of endothelial ICAM-1 to caveola- and F-actin-rich domains.</a> Nat. Cell Biol. 8, 113-123 (2006).</li><li>Schulte, D., Kuppers, V., Dartsch, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stabilizing+the+VE-cadherin-catenin+complex+blocks+leukocyte+extravasation+and+vascular+permeability.">Stabilizing the VE-cadherin-catenin complex blocks leukocyte extravasation and vascular permeability.</a> EMBO J. 30, 4157-4170 (2011).</li><li>Buul, J. D., Mul, F. P., van der Schoot, C. E., Hordijk, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ICAM-3+activation+modulates+cell-cell+contacts+of+human+bone+marrow+endothelial+cells.">ICAM-3 activation modulates cell-cell contacts of human bone marrow endothelial cells.</a> J. Vasc. Res. 41, 28-37 (2004).</li><li>Shaw, S. K., Bamba, P. S., Perkins, B. N., Luscinskas, F. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+imaging+of+vascular+endothelial-cadherin+during+leukocyte+transmigration+across+endothelium.">Real-time imaging of vascular endothelial-cadherin during leukocyte transmigration across endothelium.</a> J. Immunol. 167, 2323-2330 (2001).</li><li>Su, W. H., Chen, H., Jen, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+movements+of+VE-cadherin+and+PECAM-1+during+transmigration+of+polymorphonuclear+leukocytes+through+human+umbilical+vein+endothelium.">Differential movements of VE-cadherin and PECAM-1 during transmigration of polymorphonuclear leukocytes through human umbilical vein endothelium.</a> Blood. 100, 3597-3603 (2002).</li><li>Paulsson, J. M., Jacobson, S. H., Lundahl, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+activation+during+transmigration+in+vivo+and+in+vitro:+A+translational+study+using+the+skin+chamber+model.">Neutrophil activation during transmigration in vivo and in vitro: A translational study using the skin chamber model.</a> J. Immunol. Methods. 361, 82-88 (2010).</li><li>Williams, M. R., Azcutia, V., Newton, G., Alcaide, P., Luscinskas, F. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+mechanisms+of+neutrophil+recruitment+across+endothelium.">Emerging mechanisms of neutrophil recruitment across endothelium.</a> Trends Immunol. 32, 461-469 (2011).</li></ol>

The authors have no competing financial interests.

For this protocol, it is critical to prevent the formation of air bubbles in the flow-chambers, since this will result in cell apoptosis and a disrupted monolayer. To avoid this, we wish to stress to pay particular interest to steps 4.2 and 4.5, where the tubes are connected to the flow-chamber. Another critical step in the protocol is the priming of the PMNs that are kept at RT by incubating them for 15-30 min at 37 &#176;C prior to injecting them to the flow-chamber (step 4.1). This results in priming of leukocyte integrins, allowing them to adhere to adhesion molecules such as ICAM-1 or VCAM-1 at the endothelium.
This protocol is not restricted to study the transmigration of neutrophils. Also other leukocyte types such as monocytes or lymphocytes may be used. Note that step 4.1, i.e., priming the leukocytes may differ between leukocyte types. Also, other types of endothelial cells can be used. For this it remains critical to stimulate the endothelial cells with appropriate inflammatory stimuli such as TNF-&#945; or IL-1&#946;. If neutrophils do not respond in transmigrating, one may consider treating the neutrophils briefly (5 min) with N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) peptide16. This will stimulate the neutrophils, in particular their integrins, even further, making them more prone to adhere to the endothelium.
Typically 1 dyn/cm2 flow speed is used. This flow speed is measured in post-capillary venules, sites where most leukocyte transendothelial migration occurs17. Using the described flow-chambers, it is possible to increase flow speed up to 10 dyn/cm2. However, it is not recommended to increase the shear further. It may result in unwanted leakage of the tubing and detachment of the cells from the flow-chamber.
The results described in Figure 3 indicate that these antibodies can be used to visualize and study the dynamics of endothelial cell-cell junctions during leukocyte diapedesis. In particular, in addition to the existing transmigration assays this protocol allows to discriminate paracellular from transcellular migration under physiological flow conditions in real-time. Since the antibodies stain the cell-cell junctions, one can score the number of leukocytes that cross VE-cadherin/PECAM-1-positive junctions versus nonpositive VE-cadherin/PECAM-1 sites. This way, transcellular migration can be discriminated from paracellular migration.
It is important to underscore that these antibodies do not interfere with the function of the proteins they bind to: the PECAM-1 antibody used in this study is directed against the second extracellular domain. Endothelial cells that were plated in the presence of the antibody did not show any defects in spreading or forming a monolayer, suggesting that the antibody at least did not interfere with homotypic interactions between endothelial cells. in addition to that, both antibodies do not impair the ability of neutrophils to migrate through the endothelial monolayer. Additionally, the number of neutrophils that transmigrate across the endothelial monolayer in the absence or presence of the antibodies is not altered (data not shown). Importantly, confocal laser scanning microscopy gives us the possibility to record different fluorescent channels and DIC simultaneously.
Thus, this assay allows studying the dynamics of VE-cadherin and PECAM-1 at the same time when a neutrophil crosses the endothelial cell-cell junction and will help to understand why leukocytes choose one route over the other, i.e., paracellular versus transcellular.

Efficient and tightly controlled leukocyte transendothelial migration (TEM) is of key importance in physiological processes such as immune surveillance and acute inflammation. However, under certain patho-physiological conditions, uncontrolled and excessive TEM is observed resulting in chronic inflammatory diseases (e.g., rheumatoid arthritis, atherosclerosis, asthma). Also during tumor cell metastasis, the process of transendothelial migration is instrumental for tumor cells to leave the circulation to metastasize1-3. In order to specifically interfere with excessive leukocyte or tumor cell TEM, a detailed understanding of the regulation of this process is required.
It is believed that the TEM process occurs through different steps. Seminal studies, reviewed two decades ago by Butcher and Springer, led to the multistep model describing the process of TEM4,5. This model still holds true, although some additional steps have been included6.&#160;Alon et al.&#160;described the need for the presence of immobilized chemokines on the surface of the endothelium7. Recently, they showed that the endothelium itself generates chemokines which are presented at the endothelial apical surface8. Moreover, the same group put forward the importance of flow conditions during TEM7. Recently, several publications focused on the two different routes leukocytes can take at the final diapedesis stage of TEM. They can either go through the cell-cell junctions, i.e.,&#160;the paracellular migration route, or cross through the endothelial cell body, known as the transcellular migration route9. Carman and colleagues studied these pathways in detail and concluded that leukocytes preferentially choose the paracellular migration route (90%) over the transcellular route (10%) when crossing an umbilical vein endothelial monolayer10. However, when endothelial cells from other origins were used, e.g., brain or microvasculature, more leukocytes used the transcellular route (30%)11. The Vestweber group recently showed that when the cell-cell junctions were unable to dissociate from each other by using an knock-in animal model, replacing endogenous VE-cadherin for a VE-cadherin-alpha-catenin chimera, leukocyte TEM was fully blocked12. Surprisingly, the authors noticed that TEM was blocked in several, but not all, tissues. Overall, these elegant experiments indicated that leukocytes preferred the paracellular route over the transcellular route, although the regulatory signals that trigger this decision are still unknown.
Even though the majority of the leukocytes prefer the paracellular migration route, it is still difficult to discriminate between both pathways. In addition to that, despite several studies focusing on the role of the endothelial cell-cell junctions, the dynamics of these junctions, in particular the junctional proteins VE-cadherin and PECAM-1, during leukocyte crossing is still under debate. We developed a relatively simple assay in which these junction molecules can be monitored in real-time during leukocyte diapedesis under physiological flow conditions using fluorescently-labeled antibodies. These antibodies did not interfere with or block junctional integrity or mobility of the targeted proteins. This assay allows us to follow the dynamics of junctional proteins during the process of paracellular TEM. Additionally, this assay also allows discriminating between the para- and transcellular migration routes.

<table><tbody><tr><td>&amp;mu;-Slide VI</td><td>IBIDI</td><td>80606</td><td>Flow-chamber</td></tr><tr><td>0.45 &amp;mu;m filter&amp;nbsp;</td><td>Whatman/GE Lifesciences</td><td>10462100</td><td></td></tr><tr><td>1 ml Syringe&amp;nbsp;</td><td>BD Plastipak</td><td>300013</td><td></td></tr><tr><td>20 ml Syringe&amp;nbsp;</td><td>BD Discardit II</td><td>366296</td><td></td></tr><tr><td>21 G Needle&amp;nbsp;</td><td>BD Microlance</td><td>301155</td><td></td></tr><tr><td>Albumin&amp;nbsp;</td><td>Sanquin</td><td>15522644</td><td></td></tr><tr><td>Ammonium chloride (NH&lt;sub&gt;4&lt;/sub&gt;Cl)</td><td>Merck</td><td>1009245000</td><td></td></tr><tr><td>Calcium chloride (CaCl&lt;sub&gt;2&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>449709</td><td></td></tr><tr><td>EBM-2 Basal medium + EGM-2 SingleQuot Kit Suppl. &amp;amp; Growth Factors</td><td>Lonza</td><td>CC-3156 + CC-4176</td><td>media</td></tr><tr><td>EDTA (Titriplex III)</td><td>Merck</td><td>1370041000</td><td></td></tr><tr><td>Falcon tubes&amp;nbsp;</td><td>Corning Life Sciences</td><td>352096</td><td></td></tr><tr><td>Fibronectin</td><td>Sigma-Aldrich</td><td>F1141-2MG</td><td>FN</td></tr><tr><td>Glucose</td><td>Sigma-Aldrich</td><td>G7528</td><td></td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H3375</td><td></td></tr><tr><td>In-line Luer injection port&amp;nbsp;</td><td>IBIDI</td><td>10820</td><td></td></tr><tr><td>Magnesium sulfate heptahydrate (MgSO&lt;sub&gt;4&lt;/sub&gt;&amp;bull;7H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>Merck</td><td>105886</td><td></td></tr><tr><td>PECAM-1-ALEXA-647&amp;nbsp;</td><td>BD Pharmingen</td><td>561654</td><td>clone WM59</td></tr><tr><td>Percoll&amp;nbsp;</td><td>GE Healthcare Life Sciences</td><td>17-0891-09</td><td></td></tr><tr><td>Phosphate Buffered Saline</td><td>Fresenius Kabi&amp;nbsp; Nederland</td><td>M090001/01NL</td><td>PBS</td></tr><tr><td>Potassium chloride (KCl)</td><td>Sigma-Aldrich</td><td>P9541</td><td></td></tr><tr><td>Potassium hydrogen carbonate (KHCO&lt;sub&gt;3&lt;/sub&gt;)</td><td>Merck</td><td>1048540500</td><td></td></tr><tr><td>Potassium phosphate dibasic trihydrate (K&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>P5504</td><td></td></tr><tr><td>Silicone Tygon 3350 tubing</td><td>VWR</td><td>228-4331</td><td>tubing</td></tr><tr><td>Sodium chloride (NaCl)</td><td>Calbiochem (Millipore)</td><td>567441</td><td></td></tr><tr><td>Syringe pump&amp;nbsp;</td><td>Harvard Apparatus</td><td>model number 55-5920</td><td></td></tr><tr><td>TNF-&amp;alpha;&amp;nbsp;</td><td>Peprotech</td><td>300-01A</td><td>tumor necrosis factor</td></tr><tr><td>Trisodium citrate&amp;nbsp;</td><td>Merck</td><td>1.06447.5000</td><td>TNC</td></tr><tr><td>Vacuettes</td><td>Greiner, Germany</td><td>980044</td><td></td></tr><tr><td>VE-Cadherin-FITC&amp;nbsp;</td><td>BD Pharmingen</td><td>560411</td><td>clone 55-7H1</td></tr><tr><td>Zeiss LSM510 META&amp;nbsp;</td><td>Carl Zeiss MicroImaging, Jena, Germany</td><td></td><td></td></tr><tr><td>Zen Software 2008</td><td>Carl Zeiss MicroImaging, Jena, Germany</td><td></td><td></td></tr></tbody></table>

real-time imaging of endothelial cell-cell junctions during neutrophil transmigration under physiological flow

During inflammation, leukocytes leave the circulation and cross the endothelium to fight invading pathogens in underlying tissues. This process is known as leukocyte transendothelial migration. Two routes for leukocytes to cross the endothelial monolayer have been described: the paracellular route, i.e., through the cell-cell junctions and the transcellular route, i.e., through the endothelial cell body. However, it has been technically difficult to discriminate between the para- and transcellular route. We developed a simple in vitro assay to study the distribution of endogenous VE-cadherin and PECAM-1 during neutrophil transendothelial migration under physiological flow conditions. Prior to neutrophil perfusion, endothelial cells were briefly treated with fluorescently-labeled antibodies against VE-cadherin and PECAM-1. These antibodies did not interfere with the function of both proteins, as was determined by electrical cell-substrate impedance sensing and FRAP measurements. Using this assay, we were able to follow the distribution of endogenous VE-cadherin and PECAM-1 during transendothelial migration under flow conditions and discriminate between the para- and transcellular migration routes of the leukocytes across the endothelium.

We first tested if the antibodies did not interfere with the barrier function of the endothelium. We measured the resistance of endothelial monolayers by using electrical cell-substrate impedance sensing (ECIS). For details, see Van Buul et al.13&#160;No change in resistance was observed when the anti-VE-cadherin fluorescently-labeled antibody was added to the cells (Figure 2A). An anti-VE-cadherin antibody that is well-recognized to block VE-cadherin function reduced the resistance dramatically (Figure 2A). Also, the antibodies used for imaging do not alter the dynamics of VE-cadherin, as was assessed by measuring fluorescent recovery after photo-bleaching (FRAP) of VE-cadherin-GFP (Figure 2B).
After 1-2 min, neutrophils adhered to the activated endothelial monolayer as could be visualized in the DIC channel (Figure 3A). After crawling for 5-30 sec, the vast majority of neutrophils transmigrated through the endothelial monolayer through the cell-cell junctions that were labeled with the antibodies directed against PECAM-1 and VE-cadherin. During the process of diapedesis, the distribution of VE-cadherin and PECAM-1 was followed in real-time (Figure 3B). At sites of neutrophil diapedesis, VE-cadherin was locally disrupted and PECAM-1 showed a more ring-like structure. After completion of diapedesis, the junctions close and VE-cadherin and PECAM-1 clearly relocated at the sites of diapedesis (Figure 3C). Note that parts of the anti-PECAM-1 antibody were detected on the surface of the neutrophil once the neutrophil reached the baso-lateral side of the endothelium. &#160;However, this did not prevent&#160;neutrophils from crossing the endothelium. The observed dynamics of VE-cadherin in real-time during neutrophil transendothelial migration were in agreement with the work by Shaw and colleagues who showed, using confocal microscopy and VE-cadherin-GFP-transfected endothelial cells, that VE-cadherin-GFP diffused laterally when leukocytes crossed the endothelial cell-cell junctions14.&#160;Also work by Su and coworkers underscored our observations of PECAM-1. They showed that, next to lateral VE-cadherin diffusion upon leukocyte passage, PECAM-1 was locally redistributed into a ring around the transmigrating neutrophil15.&#160;
<img alt="Figure 1" fo:content-width="4in" src="/files/ftp_upload/51766/51766fig1highres.jpg" width="400" /> 
Figure 1.&#160;In vitro flow chamber. (A) Arrow indicates the reservoir from which the medium needs to be refreshed. (B) Silicone tubing that connects the flow chamber with the in-line Luer injection port used for injecting PMNs without disconnecting the tubing (arrowhead). (C) Connection of an empty flow-chamber&#160;to the reservoir flask filled with 37 &#176;C flow-buffer (arrowhead). (D) Connection of the flow-chamber with the TNF-&#945;-treated HUVECs to the flow-buffer-containing tubing and placed in the microscope stage (arrowhead).
<img alt="Figure 2" fo:content-width="5in" src="/files/ftp_upload/51766/51766fig2highres.jpg" width="500" /> 
Figure 2.&#160;VE-cadherin antibodies do not interfere with junctional dynamics or function. (A) Endothelial cell monolayer impedance is measured using ECIS. Y-axis expresses impedance in Ohms and x-axis represents time in hours.&#160;VE-cadherin antibody clone 55-7H1, labeled with ALEXA647 (blue line) or isotype IgG-ALEXA647 control (red line) do not alter endothelial cell monolayer impedance, whereas the VE-cadherin blocking antibody cl75 (black line) did reduce the impedance.&#160;(B) VE-cadherin-GFP was expressed in HUVEC and FRAP analysis using confocal microscopy revealed no change in fluorescence recovery in the presence or absence of the VE-cadherin 55-7H1 antibody.
<img alt="Figure 3" fo:content-width="5in" src="/files/ftp_upload/51766/51766fig3highres.jpg" width="500" /> 
Figure 3. VE-cadherin and PECAM-1 distribution during neutrophil TEM in real-time. (A) Neutrophil (marked with white line) adhering on the endothelium and crossing the cell-cell junction without affecting the distribution of VE-cadherin and PECAM-1. (B) Neutrophil crossing the endothelial monolayer through the cell-cell junctions. A local dispersion of VE-cadherin and PECAM-1 can be observed when a neutrophil protrudes through the cell-cell junctions. White line illustrates neutrophil presence still on top of the endothelium. Yellow line shows neutrophil membrane that is already underneath endothelium. (C) Neutrophil has fully crossed the endothelial monolayer. VE-cadherin and PECAM-1 are relocated at sites of diapedesis. Yellow line illustrates borders of the transmigrated neutrophil. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51766/51766fig3highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Real-time Imaging of Endothelial Cell-cell Junctions During Neutrophil Transmigration Under Physiological Flow at JoVE.com

Real-time Imaging of Endothelial Cell-cell Junctions During Neutrophil Transmigration Under Physiological Flow

Leukocytes cross the endothelial monolayer using the paracellular or the transcellular route. We developed a simple assay to follow the distribution of endogenous junctional VE-cadherin and PECAM-1 during leukocyte transendothelial migration under physiological flow to discriminate between the two transmigration routes.

During inflammation, leukocytes leave the circulation and cross the endothelium to fight invading pathogens in underlying tissues. This process is known ...

real-time-imaging-endothelial-cell-cell-junctions-during-neutrophil

Nanyang Technological University

Research

Education

JoVE Journal

JoVE Core

Cell Biology

Immunology and Infection

Unit 1

Cell-Cell Interactions

SCIENTISTS IN ACTION

19.5K Views.  Sanquin Research and Landsteiner Laboratory, AMC at University of Amsterdam. Leukocytes cross the endothelial monolayer using the paracellular or the transcellular route. We developed a simple assay to follow the distribution of endogenous junctional VE-cadherin and PECAM-1 during leukocyte transendothelial migration under physiological flow to discriminate between the two transmigration routes.

Video: Real-time Imaging of Endothelial Cell-cell Junctions During Neutrophil Transmigration Under Physiological Flow

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists. The value of 2P microscopy is that it provides rich spatiotemporal information regarding cell behaviors within intact tissues and in live mice. Leukocyte recruitment plays a significant role in host defense against infection and when unchecked, can contribute to inflammatory and autoimmune diseases. Studying leukocyte recruitment in vivo is technically challenging since cells are moving rapidly within vessels located deep within light scattering tissues. To date, most intravital imaging studies require surgical preparation to expose the blood vessels and tissues. To avoid the tissue damage and inflammation induced by surgery itself, here, we describe a non-invasive single-cell imaging approach that can be used to study leukocyte trafficking in the mouse footpad and phalanges. We discuss the technical aspects of our 2P imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Here, we describe a non-invasive two-photon (2P) microscopy approach to study leukocyte homing in the mouse footpad. We discuss the technical aspects of our tissue imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists.  The value of 2P microscopy is that it ...

Imaging Leukocyte Adhesion to the Vascular Endothelium at High Intraluminal Pressure

Worldwide, hypertension is reported to be in approximately a quarter of the population and is the leading biomedical risk factor for mortality worldwide. In the vasculature hypertension is associated with endothelial dysfunction and increased inflammation leading to atherosclerosis and various disease states such as chronic kidney disease2, stroke3 and heart failure4. An initial step in vascular inflammation leading to atherogenesis is the adhesion cascade which involves the rolling, tethering, adherence and subsequent transmigration of leukocytes through the endothelium. Recruitment and accumulation of leukocytes to the endothelium is mediated by an upregulation of adhesion molecules such as vascular cell adhesion molecule-1 (VCAM-1), intracellular cell adhesion molecule-1 (ICAM-1) and E-selectin as well as increases in cytokine and chemokine release and an upregulation of reactive oxygen species5. In vitro methods such as static adhesion assays help to determine mechanisms involved in cell-to-cell adhesion as well as the analysis of cell adhesion molecules. Methods employed in previous in vitro studies have demonstrated that acute increases in pressure on the endothelium can lead to monocyte adhesion, an upregulation of adhesion molecules and inflammatory markers6 however, similar to many in vitro assays, these findings have not been performed in real time under physiological flow conditions, nor with whole blood. Therefore, in vivo assays are increasingly utilised in animal models to demonstrate vascular inflammation and plaque development. Intravital microscopy is now widely used to assess leukocyte adhesion, rolling, migration and transmigration7-9. When combining the effects of pressure on leukocyte to endothelial adhesion the in vivo studies are less extensive. One such study examines the real time effects of flow and shear on arterial growth and remodelling but inflammatory markers were only assessed via immunohistochemistry10. Here we present a model for recording leukocyte adhesion in real time in intact pressurised blood vessels using whole blood perfusion. The methodology is a modification of an ex vivo vessel chamber perfusion model9 which enables real-time analysis of leukocyte -endothelial adhesive interactions in intact vessels. Our modification enables the manipulation of the intraluminal pressure up to 200 mmHg allowing for study not only under physiological flow conditions but also pressure conditions. While pressure myography systems have been previously demonstrated to observe vessel wall and lumen diameter11 as well as vessel contraction this is the first time demonstrating leukocyte-endothelial interactions in real time. Here we demonstrate the technique using carotid arteries harvested from rats and cannulated to a custom-made flow chamber coupled to a fluorescent microscope. The vessel chamber is equipped with a large bottom coverglass allowing a large diameter objective lens with short working distance to image the vessel. Furthermore, selected agonist and/or antagonists can be utilized to further investigate the mechanisms controlling cell adhesion. Advantages of this method over intravital microscopy include no involvement of invasive surgery and therefore a higher throughput can be obtained. This method also enables the use of localised inhibitor treatment to the desired vessel whereas intravital only enables systemic inhibitor treatment.

This is a method to visualise leukocyte adhesion to the endothelium in harvested pressurised vessels. The technique enables studying vascular adhesion under shear flow with differing intraluminal pressures up to 200 mmHg thus mimic-ing the pathophysiological conditions of high blood pressure.

Worldwide, hypertension is reported to be in approximately a quarter of the population and is the leading biomedical risk factor for mortality worldwide. ...

Tracking Neutrophil Intraluminal Crawling, Transendothelial Migration and Chemotaxis in Tissue by Intravital Video Microscopy

The recruitment of circulating leukocytes from blood stream to the inflamed tissue is a crucial and complex process of inflammation1,2. In the postcapillary venules of inflamed tissue, leukocytes initially tether and roll on the luminal surface of venular wall. Rolling leukocytes arrest on endothelium and undergo firm adhesion in response to chemokine or other chemoattractants on the venular surface. Many adherent leukocytes relocate from the initial site of adhesion to the junctional extravasation site in endothelium, a process termed intraluminal crawling3. Following crawling, leukocytes move across endothelium (transmigration) and migrate in extravascular tissue toward the source of chemoattractant (chemotaxis)4. Intravital microscopy is a powerful tool for visualizing leukocyte-endothelial cell interactions in vivo and revealing cellular and molecular mechanisms of leukocyte recruitment2,5. In this report, we provide a comprehensive description of using brightfield intravital microscopy to visualize and determine the detailed processes of neutrophil recruitment in mouse cremaster muscle in response to the gradient of a neutrophil chemoattractant. To induce neutrophil recruitment, a small piece of agarose gel (~1-mm3 size) containing neutrophil chemoattractant MIP-2 (CXCL2, a CXC chemokine) or WKYMVm (Trp-Lys-Tyr-Val-D-Met, a synthetic analog of bacterial peptide) is placed on the muscle tissue adjacent to the observed postcapillary venule. With time-lapsed video photography and computer software ImageJ, neutrophil intraluminal crawling on endothelium, neutrophil transendothelial migration and the migration and chemotaxis in tissue are visualized and tracked. This protocol allows reliable and quantitative analysis of many neutrophil recruitment parameters such as intraluminal crawling velocity, transmigration time, detachment time, migration velocity, chemotaxis velocity and chemotaxis index in tissue. We demonstrate that using this protocol, these neutrophil recruitment parameters can be stably determined and the single cell locomotion conveniently tracked in vivo.

We describe a protocol of brightfield intravital microscopy for measuring dynamic neutrophil-endothelial cell interactions during neutrophil recruitment in response to the source of a neutrophil chemoattractant in vivo. Neutrophil intraluminal crawling, transendothelial migration and chemotaxis in mouse cremaster muscle tissue are visualized with time-lapsed video photography and tracked with ImageJ.

The recruitment of circulating leukocytes from blood stream to the inflamed tissue is a crucial and complex process of inflammation1,2. In the ...

Isolation of Human Umbilical Vein Endothelial Cells and Their Use in the Study of Neutrophil Transmigration Under Flow Conditions

Neutrophils are the most abundant type of white blood cell. They form an essential part of the innate immune system1. During acute inflammation, neutrophils are the first inflammatory cells to migrate to the site of injury. Recruitment of neutrophils to an injury site is a stepwise process that includes first, dilation of blood vessels to increase blood flow; second, microvascular structural changes and escape of plasma proteins from the bloodstream; third, rolling, adhesion and transmigration of the neutrophil across the endothelium; and fourth accumulation of neutrophils at the site of injury2,3. A wide array of in vivo and in vitro methods has evolved to enable the study of these processes4. This method focuses on neutrophil transmigration across human endothelial cells.
One popular method for examining the molecular processes involved in neutrophil transmigration utilizes human neutrophils interacting with primary human umbilical vein endothelial cells (HUVEC)5. Neutrophil isolation has been described visually elsewhere6; thus this article will show the method for isolation of HUVEC. Once isolated and grown to confluence, endothelial cells are activated resulting in the upregulation of adhesion and activation molecules. For example, activation of endothelial cells with cytokines like TNF-&alpha; results in increased E-selectin and IL-8 expression7. E-selectin mediates capture and rolling of neutrophils and IL-8 mediates activation and firm adhesion of neutrophils. After adhesion neutrophils transmigrate. Transmigration can occur paracellularly (through endothelial cell junctions) or transcellularly (through the endothelial cell itself). In most cases, these interactions occur under flow conditions found in the vasculature7,8.
The parallel plate flow chamber is a widely used system that mimics the hydrodynamic shear stresses found in vivo and enables the study of neutrophil recruitment under flow condition in vitro9,10. Several companies produce parallel plate flow chambers and each have advantages and disadvantages. If fluorescent imaging is needed, glass or an optically similar polymer needs to be used. Endothelial cells do not grow well on glass.
Here we present an easy and rapid method for phase-contrast, DIC and fluorescent imaging of neutrophil transmigration using a low volume ibidi channel slide made of a polymer that supports the rapid adhesion and growth of human endothelial cells and has optical qualities that are comparable to glass. In this method, endothelial cells were grown and stimulated in an ibidi &mu;slide. Neutrophils were introduced under flow conditions and transmigration was assessed. Fluorescent imaging of the junctions enabled real-time determination of the extent of paracellular versus transcellular transmigration.

This article first describes a procedure for isolating human endothelial cells from umbilical veins and then shows how to use these cells to examine neutrophil transmigration under flow conditions. By using a low-volume flow chamber made from a polymer with the optical characteristics of glass, live-cell fluorescent imaging of rare cell populations is also possible.

Neutrophils are the most abundant type of white blood cell. They form an essential part of the innate immune system1. During acute inflammation, ...

Real-time Imaging of Heterotypic Platelet-neutrophil Interactions on the Activated Endothelium During Vascular Inflammation and Thrombus Formation in Live Mice

Interaction of activated platelets and leukocytes (mainly neutrophils) on the activated endothelium mediates thrombosis and vascular inflammation.1,2 During thrombus formation at the site of arteriolar injury, platelets adherent to the activated endothelium and subendothelial matrix proteins support neutrophil rolling and adhesion.3 Conversely, under venular inflammatory conditions, neutrophils adherent to the activated endothelium can support adhesion and accumulation of circulating platelets. Heterotypic platelet-neutrophil aggregation requires sequential processes by the specific receptor-counter receptor interactions between cells.4 It is known that activated endothelial cells release adhesion molecules such as von Willebrand factor, thereby initiating platelet adhesion and accumulation under high shear conditions.5 Also, activated endothelial cells support neutrophil rolling and adhesion by expressing selectins and intercellular adhesion molecule-1 (ICAM-1), respectively, under low shear conditions.4 Platelet P-selectin interacts with neutrophils through P-selectin glycoprotein ligand-1 (PSGL-1), thereby inducing activation of neutrophil &beta;2 integrins and firm adhesion between two cell types. Despite the advances in in vitro experiments in which heterotypic platelet-neutrophil interactions are determined in whole blood or isolated cells,6,7 those studies cannot manipulate oxidant stress conditions during vascular disease. In this report, using fluorescently-labeled, specific antibodies against a mouse platelet and neutrophil marker, we describe a detailed intravital microscopic protocol to monitor heterotypic interactions of platelets and neutrophils on the activated endothelium during TNF-&alpha;-induced inflammation or following laser-induced injury in cremaster muscle microvessels of live mice.

Here we report an experimental technique of fluorescence intravital microscopy to visualize heterotypic platelet-neutrophil interactions on the activated endothelium during vascular inflammation and thrombus formation in live mice. This microscopic technology will be valuable to study the molecular mechanism of vascular disease and to test pharmacologic agents under pathophysiological conditions.

Interaction of activated platelets and leukocytes (mainly neutrophils) on the activated endothelium mediates thrombosis and vascular inflammation.1,2 ...

An Endothelial Planar Cell Model for Imaging Immunological Synapse Dynamics

Adaptive immunity is regulated by dynamic interactions between T cells and antigen presenting cells (&#39;APCs&#39;) referred to as &#39;immunological synapses&#39;. Within these intimate cell-cell interfaces discrete sub-cellular clusters of MHC/Ag-TCR, F-actin, adhesion and signaling molecules form and remodel rapidly. These dynamics are thought to be critical determinants of both the efficiency and quality of the immune responses that develop and therefore of protective versus pathologic immunity. Current understanding of immunological synapses with physiologic APCs is limited by the inadequacy of the obtainable imaging resolution. Though artificial substrate models (e.g., planar lipid bilayers) offer excellent resolution and have been extremely valuable tools, they are inherently non-physiologic and oversimplified. Vascular and lymphatic endothelial cells have emerged as an important peripheral tissue (or stromal) compartment of &#39;semi-professional APCs&#39;. These APCs (which express most of the molecular machinery of professional APCs) have the unique feature of forming virtually planar cell surface and are readily transfectable (e.g., with fluorescent protein reporters). Herein a basic approach to implement endothelial cells as a novel and physiologic &#39;planar cellular APC model&#39; for improved imaging and interrogation of fundamental antigenic signaling processes will be described.

Adaptive immunity is controlled by dynamic 'immunological synapses' formed between T cells and antigen presenting cells. This protocol describes methods for investigating endothelial cells both as understudied physiologic APCs and as a novel type of 'planar cellular APC model'.

Adaptive immunity is regulated by dynamic interactions between T cells and antigen presenting cells (&#39;APCs&#39;) referred to as &#39;immunological ...

Imaging Neutrophils and Monocytes in Mesenteric Veins by Intravital Microscopy on Anaesthetized Mice in Real Time

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in vivo is crucial for understanding the molecular basis of leukocyte transendothelial migration and interaction with vascular endothelium. One powerful approach involves intravital microscopy on transgenic mice expressing fluorescent proteins in cells of interest.
Here we present a protocol for imaging monocytes and neutrophils in the CX3CR1gfp/wt mouse i.v. injected with orange dye-labeled neutrophils with an inverted confocal microscope. Time-lapse movies gathered from 30 min&#160;to several hours of imaging allow the analysis of leukocyte behavior in mesenteric veins under both steady state and inflammatory conditions. We also describe the steps to locally induce blood vessel inflammation with TLR2/TLR1 agonist Pam3SK4 and monitor the subsequent recruitment of neutrophils and monocytes.
The presented technique can also be used to monitor other populations of leukocytes and investigate molecules implicated in leukocyte recruitment or trafficking using other stimuli or transgenic mice.

We detail a protocol to monitor the behavior of neutrophils and monocytes in mesenteric veins under steady state and inflammatory conditions using intravital confocal microscopy on anaesthetized mice.

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in ...

Study of the Actin Cytoskeleton in Live Endothelial Cells Expressing GFP-Actin

The microvascular endothelium plays an important role as a selectively permeable barrier to fluids and solutes. The adhesive junctions between endothelial cells regulate permeability of the endothelium, and many studies have indicated the important contribution of the actin cytoskeleton to determining junctional integrity1-5. A cortical actin belt is thought to be important for the maintenance of stable junctions1, 2, 4, 5. In contrast, actin stress fibers are thought to generate centripetal tension within endothelial cells that weakens junctions2-5. Much of this theory has been based on studies in which endothelial cells are treated with inflammatory mediators known to increase endothelial permeability, and then fixing the cells and labeling F-actin for microscopic observation. However, these studies provide a very limited understanding of the role of the actin cytoskeleton because images of fixed cells provide only snapshots in time with no information about the dynamics of actin structures5. 
Live-cell imaging allows incorporation of the dynamic nature of the actin cytoskeleton into the studies of the mechanisms determining endothelial barrier integrity. A major advantage of this method is that the impact of various inflammatory stimuli on actin structures in endothelial cells can be assessed in the same set of living cells before and after treatment, removing potential bias that may occur when observing fixed specimens. Human umbilical vein endothelial cells (HUVEC) are transfected with a GFP-&beta;-actin plasmid and grown to confluence on glass coverslips. Time-lapse images of GFP-actin in confluent HUVEC are captured before and after the addition of inflammatory mediators that elicit time-dependent changes in endothelial barrier integrity. These studies enable visual observation of the fluid sequence of changes in the actin cytoskeleton that contribute to endothelial barrier disruption and restoration. 
Our results consistently show local, actin-rich lamellipodia formation and turnover in endothelial cells. The formation and movement of actin stress fibers can also be observed. An analysis of the frequency of formation and turnover of the local lamellipodia, before and after treatment with inflammatory stimuli can be documented by kymograph analyses. These studies provide important information on the dynamic nature of the actin cytoskeleton in endothelial cells that can used to discover previously unidentified molecular mechanisms important for the maintenance of endothelial barrier integrity.

Microscopic imaging of live endothelial cells expressing GFP-actin allows characterization of dynamic changes in cytoskeletal structures. Unlike techniques that use fixed specimens, this method provides a detailed assessment of temporal changes in the actin cytoskeleton in the same cells before, during, and after various physical, pharmacological, or inflammatory stimuli.

The microvascular endothelium plays an important role as a selectively permeable barrier to fluids and solutes. The adhesive junctions between endothelial ...

Biology

Simultaneous Study of the Recruitment of Monocyte Subpopulations Under Flow In Vitro

The recruitment of monocytes from the blood to targeted peripheral tissues is critical to the inflammatory process during tissue injury, tumor development and autoimmune diseases. This is facilitated through a process of capture from free flow onto the luminal surface of activated endothelial cells, followed by their adhesion and transendothelial migration (transmigration) into the underlying affected tissue. However, the mechanisms that support the preferential and context-dependent recruitment of monocyte subpopulations are still not fully understood. Therefore, we have developed a method that allows the recruitment of different monocyte subpopulations to be simultaneously visualized and measured under flow. This method, based on time-lapse confocal imaging, allows for the unambiguous distinction between adherent and transmigrated monocytes. Here, we describe how this method can be used to simultaneously study the recruitment cascade of pro-angiogenic and non-angiogenic monocytes in vitro. Furthermore, this method can be extended to study the different steps of recruitment of up to three monocyte populations.

Here, we present an integrated protocol that measures monocyte subpopulation trafficking under flow in vitro by use of specific surface markers and confocal fluorescence microscopy. This protocol can be used to explore sequential recruitment steps as well as to profile other leukocyte subtypes using other specific surface markers.

The recruitment of monocytes from the blood to targeted peripheral tissues is critical to the inflammatory process during tissue injury, tumor development ...

A Microphysiological System to Study Leukocyte-Endothelial Cell Interaction during Inflammation

Leukocyte-endothelial cell interactions play an important role in inflammatory diseases such as sepsis. During inflammation, excessive migration of activated leukocytes across the vascular endothelium into key organs can lead to organ failure. A physiologically relevant biomimetic microfluidic assay (bMFA) has been developed and validated using several experimental and computational techniques, which can reproduce the entire leukocyte rolling/adhesion/migration cascade to study leukocyte-endothelial cell interactions. Microvascular networks obtained from in vivo images in rodents were digitized using a Geographic Information System (GIS) approach and microfabricated with polydimethylsiloxane (PDMS) on a microscope slide. To study the effect of shear rate and vascular topology on leukocyte-endothelial cell interactions, a Computational Fluid Dynamics (CFD) model was developed to generate a corresponding map of shear rates and velocities throughout the network. The bMFA enables the quantification of leukocyte-endothelial cells interactions, including rolling velocity, number of adhered leukocytes in response to different shear rates, number of migrated leukocytes, endothelial cell permeability, adhesion molecule expression and other important variables. Furthermore, by using human-related samples, such as human endothelial cells and leukocytes, bMFA provides a tool for rapid screening of potential therapeutics to increase their clinical translatability.

In this protocol, a biomimetic microfluid assay, which can reproduce a physiologically relevant microvascular environment and reproduce the entire leukocyte adhesion/migration cascade, is employed to study leukocyte-endothelial cell interactions in inflammatory disease.

Leukocyte-endothelial cell interactions play an important role in inflammatory diseases such as sepsis. During inflammation, excessive migration of ...

Real-time Imaging of Endothelial Cell-cell Junctions During Neutrophil Transmigration Under Physiological Flow

Summary

Explore More Videos

Chapters in this video

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Imaging Leukocyte Adhesion to the Vascular Endothelium at High Intraluminal Pressure

Tracking Neutrophil Intraluminal Crawling, Transendothelial Migration and Chemotaxis in Tissue by Intravital Video Microscopy

Isolation of Human Umbilical Vein Endothelial Cells and Their Use in the Study of Neutrophil Transmigration Under Flow Conditions

Real-time Imaging of Heterotypic Platelet-neutrophil Interactions on the Activated Endothelium During Vascular Inflammation and Thrombus Formation in Live Mice

An Endothelial Planar Cell Model for Imaging Immunological Synapse Dynamics

Imaging Neutrophils and Monocytes in Mesenteric Veins by Intravital Microscopy on Anaesthetized Mice in Real Time

Study of the Actin Cytoskeleton in Live Endothelial Cells Expressing GFP-Actin

Simultaneous Study of the Recruitment of Monocyte Subpopulations Under Flow In Vitro

A Microphysiological System to Study Leukocyte-Endothelial Cell Interaction during Inflammation