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 border="0" cellpadding="0" cellspacing="0" width="1251">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:471px;">Name of Material/ Equipment</td>
			<td style="width:365px;">Company</td>
			<td style="width:184px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
			<td style="width:64px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">&#956;-Slide VI</td>
			<td style="width:365px;">IBIDI</td>
			<td style="width:184px;">80606</td>
			<td>Flow-chamber</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">0.45 &#956;m filter&#160;</td>
			<td>Whatman/GE Lifesciences</td>
			<td style="width:184px;">10462100</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:471px;">1 mL syringe&#160;</td>
			<td style="width:365px;">BD Plastipak</td>
			<td style="width:184px;">300013</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">20 mL syringe&#160;</td>
			<td>BD Discardit II</td>
			<td style="width:184px;">366296</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:471px;">21G needle&#160;</td>
			<td style="width:365px;">BD Microlance</td>
			<td style="width:184px;">301155</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Albumin&#160;</td>
			<td style="width:365px;">Sanquin</td>
			<td style="width:184px;">15522644</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:471px;">Ammunium chloride (NH4Cl)</td>
			<td style="width:365px;">Merck</td>
			<td style="width:184px;">1009245000</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:471px;">Calcium chloride (CaCl2)</td>
			<td style="width:365px;">Sigma-Aldrich</td>
			<td style="width:184px;">449709</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:471px;">EBM-2 Basal medium + EGM-2 SingleQuot Kit Suppl. &#38; Growth Factors</td>
			<td style="width:365px;">Lonza</td>
			<td style="width:184px;">CC-3156 + CC-4176</td>
			<td>media</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:471px;">EDTA (Titriplex III)</td>
			<td style="width:365px;">Merck</td>
			<td style="width:184px;">1370041000</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Falcon tubes&#160;</td>
			<td>Corning Life Sciences</td>
			<td style="width:184px;">352096</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:471px;">Fibronectin</td>
			<td style="width:365px;">Sigma-Aldrich</td>
			<td style="width:184px;">F1141-2MG</td>
			<td>FN</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:471px;">Glucose</td>
			<td style="width:365px;">Sigma-Aldrich</td>
			<td style="width:184px;">G7528</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:471px;">HEPES</td>
			<td style="width:365px;">Sigma-Aldrich</td>
			<td style="width:184px;">H3375</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">In-line Luer Injection port&#160;</td>
			<td>IBIDI</td>
			<td style="width:184px;">10820</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:471px;">Magnesium sulfate heptahydrate (MgSO4.7H2O)</td>
			<td style="width:365px;">Merck</td>
			<td style="width:184px;">105886</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PECAM-1-ALEXA-647&#160;</td>
			<td style="width:365px;">BD Pharmingen</td>
			<td style="width:184px;">561654</td>
			<td>clone WM59 
			stock concentration 0.1mg/mL</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Percoll&#160;</td>
			<td>GE Healthcare Life Sciences</td>
			<td style="width:184px;">17-0891-09</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:471px;">Phosphate Buffered Saline</td>
			<td style="width:365px;">Fresenius Kabi&#160; Nederland</td>
			<td style="width:184px;">M090001/01NL</td>
			<td>PBS</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:471px;">Potassium chloride (KCl)</td>
			<td style="width:365px;">Sigma-Aldrich</td>
			<td style="width:184px;">P9541</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:471px;">Potassium hydrogen carbonate (KHCO3)</td>
			<td style="width:365px;">Merck</td>
			<td style="width:184px;">1048540500</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:471px;">Potassium phosphate dibasic trihydrate (K2HPO4)</td>
			<td style="width:365px;">Sigma-Aldrich</td>
			<td style="width:184px;">P5504</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:471px;">Silicone Tygon 3350 tubing</td>
			<td style="width:365px;">VWR</td>
			<td style="width:184px;">228-4331</td>
			<td>tubing</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:471px;">Sodium chloride (NaCl)</td>
			<td style="width:365px;">Calbiochem (Millipore)</td>
			<td style="width:184px;">567441</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Syringe pump&#160;</td>
			<td style="width:365px;">Harvard Apparatus</td>
			<td>model number 55-5920</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TNF&#945;&#160;</td>
			<td style="width:365px;">Peprotech</td>
			<td style="width:184px;">300-01A</td>
			<td>tumor necrosis factor</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">trisodium citrate&#160;</td>
			<td style="width:365px;">Merck</td>
			<td style="width:184px;">1.06447.5000</td>
			<td>TNC</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Vacuettes</td>
			<td style="width:365px;">Greiner, Germany</td>
			<td style="width:184px;">980044</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">VE-Cadherin-FITC&#160;</td>
			<td style="width:365px;">BD Pharmingen</td>
			<td style="width:184px;">560411</td>
			<td>clone 55-7H1</td>
			<td>stock concentration 0.5mg/mL</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Zeiss LSM510 META&#160;</td>
			<td style="width:365px;">Carl Zeiss MicroImaging, Jena, Germany</td>
			<td style="width:184px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:471px;">Zen Software 2008</td>
			<td style="width:365px;">Carl Zeiss MicroImaging, Jena, Germany</td>
			<td style="width:184px;"></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/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 ...

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19.4K 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

Real-time Imaging of Leukotriene B4 Mediated Cell Migration and BLT1 Interactions with &beta;-arrestin

G-protein coupled receptors (GPCRs) belong to the seven transmembrane protein family and mediate the transduction of extracellular signals to intracellular responses. GPCRs control diverse biological functions such as chemotaxis, intracellular calcium release, gene regulation in a ligand dependent manner via heterotrimeric G-proteins1-2. Ligand binding induces a series of conformational changes leading to activation of heterotrimeric G-proteins that modulate levels of second messengers such as cyclic adenosine monophosphate (cAMP), inositol triphosphate (IP3) and diacyl glycerol (DG). Concomitant with activation of the receptor ligand binding also initiates a series of events to attenuate the receptor signaling via desensitization, sequestration and/or internalization. The desensitization process of GPCRs occurs via receptor phosphorylation by G-protein receptor kinases (GRKs) and subsequent binding of &beta;-arrestins3. &beta;-arrestins are cytosolic proteins and translocate to membrane upon GPCR activation, binding to phosphorylated receptors (most cases) there by facilitating receptor internalization 4-6.
Leukotriene B4 (LTB4) is a pro-inflammatory lipid molecule derived from arachidonic acid pathway and mediates its actions via GPCRs, LTB4 receptor 1 (BLT1; a high affinity receptor) and LTB4 receptor 2 (BLT2; a low affinity receptor)7-9. The LTB4-BLT1 pathway has been shown to be critical in several inflammatory diseases including, asthma, arthritis and atherosclerosis10-17. The current paper describes the methodologies developed to monitor LTB4-induced leukocyte migration and the interactions of BLT1 with &beta;-arrestin and , receptor translocation in live cells using microscopy imaging techniques18-19.
Bone marrow derived dendritic cells from C57BL/6 mice were isolated and cultured as previously described 20-21. These cells were tested in live cell imaging methods to demonstrate LTB4 induced cell migration. The human BLT1 was tagged with red fluorescent protein (BLT1-RFP) at C-terminus and &beta;-arrestin1 tagged with green fluorescent protein (&beta;-arr-GFP) and transfected the both plasmids into Rat Basophilic Leukomia (RBL-2H3) cell lines18-19. The kinetics of interaction between these proteins and localization were monitored using live cell video microscopy. The methodologies in the current paper describe the use of microscopic techniques to investigate the functional responses of G-protein coupled receptors in live cells. The current paper also describes the use of Metamorph software to quantify the fluorescence intensities to determine the kinetics of receptor and cytosolic protein interactions.

Real-time Imaging of Leukotriene B4 Mediated Cell Migration and BLT1 Interactions with &amp;#946;-arrestin

This paper describes the methodology to determine the chemotactic response of leukocytes to specific ligands and identify interactions between the cell surface receptors and cytosolic proteins using live cell imaging techniques.

G-protein coupled receptors (GPCRs) belong to the seven transmembrane protein family and mediate the transduction of extracellular signals to ...

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, ...

Detection of MicroRNAs in Microglia by Real-time PCR in Normal CNS and During Neuroinflammation

Microglia are cells of the myeloid lineage that reside in the central nervous system (CNS)1. These cells play an important role in pathologies of many diseases associated with neuroinflammation such as multiple sclerosis (MS)2. Microglia in a normal CNS express macrophage marker CD11b and exhibit a resting phenotype by expressing low levels of activation markers such as CD45. During pathological events in the CNS, microglia become activated as determined by upregulation of CD45 and other markers3. The factors that affect microglia phenotype and functions in the CNS are not well studied. MicroRNAs (miRNAs) are a growing family of conserved molecules (~22 nucleotides long) that are involved in many normal physiological processes such as cell growth and differentiation4 and pathologies such as inflammation5. MiRNAs downregulate the expression of certain target genes by binding complementary sequences of their mRNAs and play an important role in the activation of innate immune cells including macrophages6 and microglia7. In order to investigate miRNA-mediated pathways that define the microglial phenotype, biological function, and to distinguish microglia from other types of macrophages, it is important to quantitatively assess the expression of particular microRNAs in distinct subsets of CNS-resident microglia. Common methods for measuring the expression of miRNAs in the CNS include quantitative PCR from whole neuronal tissue and in situ hybridization. However, quantitative PCR from whole tissue homogenate does not allow the assessment of the expression of miRNA in microglia, which represent only 5-15% of the cells of neuronal tissue. Hybridization in situ allows the assessment of the expression of microRNA in specific cell types in the tissue sections, but this method is not entirely quantitative. In this report we describe a quantitative and sensitive method for the detection of miRNA by real-time PCR in microglia isolated from normal CNS or during neuroinflammation using experimental autoimmune encephalomyelitis (EAE), a mouse model for MS. The described method will be useful to measure the level of expression of microRNAs in microglia in normal CNS or during neuroinflammation associated with various pathologies including MS, stroke, traumatic injury, Alzheimer's disease and brain tumors.

Microglia are resident macrophages that provide the first line of defense and immune surveillance of the central nervous system. MicroRNAs are regulatory molecules that play an important role in many physiological processes including activation and differentiation of macrophages. In this article, we describe the method for measurement of microRNAs in microglia.

Microglia are cells of the myeloid lineage that reside in the central nervous system (CNS)1. These cells play an important role in pathologies of many ...

Real-time Live Imaging of T-cell Signaling Complex Formation

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating the activation and responses of multiple immune cells 3,4. T-cell activation is dependent on the recognition of specific antigens displayed by antigen presenting cells (APCs). The T-cell antigen receptor (TCR) is specific to each T-cell clone and determines antigen specificity 5. The binding of the TCR to the antigen induces the phosphorylation of components of the TCR complex. In order to promote T-cell activation, this signal must be transduced from the membrane to the cytoplasm and the nucleus, initiating various crucial responses such as recruitment of signaling proteins to the TCR;APC site (the immune synapse), their molecular activation, cytoskeletal rearrangement, elevation of intracellular calcium concentration, and changes in gene expression 6,7. The correct initiation and termination of activating signals is crucial for appropriate T-cell responses. The activity of signaling proteins is dependent on the formation and termination of protein-protein interactions, post translational modifications such as protein phosphorylation, formation of protein complexes, protein ubiquitylation and the recruitment of proteins to various cellular sites 8. Understanding the inner workings of the T-cell activation process is crucial for both immunological research and clinical applications.
Various assays have been developed in order to investigate protein-protein interactions; however, biochemical assays, such as the widely used co-immunoprecipitation method, do not allow protein location to be discerned, thus precluding the observation of valuable insights into the dynamics of cellular mechanisms. Additionally, these bulk assays usually combine proteins from many different cells that might be at different stages of the investigated cellular process. This can have a detrimental effect on temporal resolution. The use of real-time imaging of live cells allows both the spatial tracking of proteins and the ability to temporally distinguish between signaling events, thus shedding light on the dynamics of the process 9,10. We present a method of real-time imaging of signaling-complex formation during T-cell activation. Primary T-cells or T-cell lines, such as Jurkat, are transfected with plasmids encoding for proteins of interest fused to monomeric fluorescent proteins, preventing non-physiological oligomerization 11. Live T cells are dropped over a coverslip pre-coated with T-cell activating antibody 8,9, which binds to the CD3/TCR complex, inducing T-cell activation while overcoming the need for specific activating antigens. Activated cells are constantly imaged with the use of confocal microscopy. Imaging data are analyzed to yield quantitative results, such as the colocalization coefficient of the signaling proteins.

We describe a live-cell imaging method that provides insight into protein dynamics during the T-cell activation process. We demonstrate the combined usage of the T-cell spreading assay, confocal microscopy and imaging analysis to yield quantitative results to follow signaling complex formation throughout T-cell activation.

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating ...

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 ...

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

The vascular endothelium plays an integral part in the inflammatory response. During the acute phase of inflammation, endothelial cells (ECs) are activated by host mediators or directly by conserved microbial components or host-derived danger molecules. Activated ECs express cytokines, chemokines and adhesion molecules that mobilize, activate and retain leukocytes at the site of infection or injury. Neutrophils are the first leukocytes to arrive, and adhere to the endothelium through a variety of adhesion molecules present on the surfaces of both cells. The main functions of neutrophils are to directly eliminate microbial threats, promote the recruitment of other leukocytes through the release of additional factors, and initiate wound repair. Therefore, their recruitment and attachment to the endothelium is a critical step in the initiation of the inflammatory response. In this report, we describe an in vitro neutrophil adhesion assay using calcein AM-labeled primary human neutrophils to quantitate the extent of microvascular endothelial cell activation under static conditions. This method has the additional advantage that the same samples quantitated by fluorescence spectrophotometry can also be visualized directly using fluorescence microscopy for a more qualitative assessment of neutrophil binding.

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

Neutrophil adherence to the activated endothelium at sites of infection is an integral component of the host's inflammatory response. Described in this report is a neutrophil binding assay that allows for the in vitro quantitation of primary human neutrophil binding to endothelial cells activated by inflammatory mediators under static conditions.

The vascular  endothelium plays an integral part in the inflammatory response. During the  acute phase of inflammation, endothelial cells (ECs) are ...

Human Neutrophil Flow Chamber Adhesion Assay

Neutrophil firm adhesion to endothelial cells plays a critical role in inflammation in both health and disease. The process of neutrophil firm adhesion involves many different adhesion molecules including members of the &beta;2 integrin family and their counter-receptors of the ICAM family. Recently, naturally occurring genetic variants in both &beta;2 integrins and ICAMs are reported to be associated with autoimmune disease. Thus, the quantitative adhesive capacity of neutrophils from individuals with varying allelic forms of these adhesion molecules is important to study in relation to mechanisms underlying development of autoimmunity. Adhesion studies in flow chamber systems can create an environment with fluid shear stress similar to that observed in the blood vessel environment in vivo. Here, we present a method using a flow chamber assay system to study the quantitative adhesive properties of human peripheral blood neutrophils to human umbilical vein endothelial cell (HUVEC) and to purified ligand substrates. With this method, the neutrophil adhesive capacities from donors with different allelic variants in adhesion receptors can be assessed and compared. This method can also be modified to assess adhesion of other primary cell types or cell lines.

A method of quantitating neutrophil adhesion is reported. This method creates a dynamic flow environment similar to that encountered in a blood vessel. It allows the investigation of neutrophil adhesion to either purified adhesion molecules (ligand) or endothelial cell substrate (HUVEC) in a context similar to the in vivo environment with sheer stress.

Neutrophil firm adhesion to endothelial cells plays a critical role in inflammation in both health and disease. The process of neutrophil firm adhesion ...

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques enable the observation of live cellular behavior inside the organ but can be challenging in some types of cancer due to organ and tumor accessibility such as intestine. Direct observation of intestinal tumors has not been previously reported. A surgical procedure described here allows direct observation of myeloid cell dynamics within the intestinal tumors in live mice by using transgenic fluorescent reporter mice and injectable tracers or antibodies. For this purpose, a four-color, multi-region, micro-lensed spinning disk confocal microscope that allows long-term continuous imaging with rapid image acquisition has been used. ApcMin/+ mice that develop multiple adenomas in the small intestine are crossed with c-fms-EGFP mice to visualize myeloid cells and with ACTB-ECFP mice to visualize intestinal epithelial cells of the crypts. Procedures for labeling different tumor components, such as blood vessels and neutrophils, and the procedure for positioning the tumor for imaging through the serosal surface are also described. Time-lapse movies compiled from several hours of imaging allow the analysis of myeloid cell behavior in situ in the intestinal microenvironment.

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

By using transgenic reporter mice and injectable fluorescent labels, long-term intravital spinning disk confocal microscopy enables direct visualization of myeloid cell behavior into intestinal adenoma in the ApcMin/+ colorectal cancer model.

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques ...

Isolation, Expansion, and Adipogenic Induction of CD34+CD31+ Endothelial Cells from Human Omental and Subcutaneous Adipose Tissue

Obesity is accompanied by an extensive remodeling of adipose tissue primarily via adipocyte hypertrophy. Extreme adipocyte growth results in a poor response to insulin, local hypoxia, and inflammation. By stimulating the differentiation of functional white adipocytes from progenitors, radical hypertrophy of the adipocyte population can be prevented and, consequently, the metabolic health of adipose tissue can be improved along with a reduction of inflammation. Also, by stimulating a differentiation of beige/brown adipocytes, the total body energy expenditure can be increased, resulting in weight loss. This approach could prevent the development of obesity co-morbidities such as type 2 diabetes and cardiovascular disease.
This paper describes the isolation, expansion, and differentiation of white and beige adipocytes from a subset of human adipose tissue endothelial cells that co-express the CD31 and CD34 markers. The method is relatively cheap and is not labor-intensive. It requires access to human adipose tissue and the subcutaneous depot is suitable for sampling. For this protocol, fresh adipose tissue samples from morbidly obese subjects [body mass index (BMI) &#62;35] are collected during bariatric surgery procedures. Using a sequential immunoseparation from the stromal vascular fraction, enough cells are produced from as little as 2&#8211;3 g of fat. These cells can be expanded in culture over 10&#8211;14 days, can be cryopreserved, and retain their adipogenic properties with passaging up to passage 5&#8211;6. The cells are treated for 14 days with an adipogenic cocktail using a combination of human insulin and the PPAR&#947; agonist-rosiglitazone.
This methodology can be used for obtaining proof of concept experiments on molecular mechanisms that drive adipogenic responses in adipose endothelial cells, or for screening new drugs that can enhance the adipogenic response directed either towards white or beige/brown adipocyte differentiation. Using small subcutaneous biopsies, this methodology can be used to screen out non-responder subjects for clinical trials aimed to stimulate beige/brown and white adipocytes for the treatment of obesity and co-morbidities.

The differentiation of white and beige adipocytes from adipose tissue vascular progenitors bears potential for metabolic improvement in obesity. We describe protocols for a CD34+CD31+ endothelial cell isolation from human fat and for a subsequent in vitro expansion and differentiation into white and beige adipocytes. Several downstream applications are discussed.

Obesity is accompanied by an extensive remodeling of adipose tissue primarily via adipocyte hypertrophy. Extreme adipocyte growth results in a poor ...

Real-time Imaging and Quantification of Fungal Biofilm Development Using a Two-Phase Recirculating Flow System

In oropharyngeal candidiasis, members of the genus Candida must adhere to and grow on the oral mucosal surface while under the effects of salivary flow. While models for the growth under flow have been developed, many of these systems are expensive, or do not allow imaging while the cells are under flow. We have developed a novel apparatus that allows us to image the growth and development of Candida albicans cells under flow and in real-time. Here, we detail the protocol for the assembly and use of this flow apparatus, as well as the quantification of data that are generated. We are able to quantify the rates that the cells attach to and detach from the slide, as well as to determine a measure of the biomass on the slide over time. This system is both economical and versatile, working with many types of light microscopes, including inexpensive benchtop microscopes, and is capable of extended imaging times compared to other flow systems. Overall, this is a low-throughput system that can provide highly detailed real-time information on the biofilm growth of fungal species under flow.

We describe the assembly, operation, and cleaning of a flow apparatus designed to image fungal biofilm formation in real time while under flow. We also provide and discuss quantitative algorithms to be used on the acquired images.

In oropharyngeal candidiasis, members of the genus Candida must adhere to and grow on the oral mucosal surface while under the effects of salivary flow. ...