We thank Drs. Martin M. Schmitt and Line Fraemohs. This work was supported by the Netherlands Foundation for Scientific Research (ZonMW VIDI 016.126.358, the Landsteiner Foundation for Blood Transfusion Research (LSBR Nr. 1638) and Deutsche Forschungsgemeinschaft (SFB1123/A2) awarded to R.R.K.

All methods described here have been approved by the Medical Ethical and Animal Ethical Boards of Maastricht University.
1. Flow-Based Assay with Human Cells
<ol>
	<li>Isolation of platelets from human blood
	<ol>
		<li>Draw venous blood in citrate (3.2%) anticoagulant.</li>
		<li>Add 1/15 volume of Acid Citrate Dextrose (ACD: 80 mM trisodium citrate, 52 mM citric acid and 183 mM glucose) to the blood.</li>
		<li>Centrifuge at 350 x g without brake for 15 min to obtain platelet rich plasma (PRP).</li>
		<li>Transfer the supernatant to a 15 mL conical tube and add 1/20 volume of ACD.</li>
		<li>Centrifuge at 1,110 g without brake for 15 min to obtain platelet poor plasma (PPP).</li>
		<li>Discard the supernatant. Cut off the end of the pipet tip (P 1000), making it wide bore, which aids in the prevention of platelet activation, and carefully suspend the platelet pellet in the same volume as PRP in platelet buffer pH 6.6 (PB: 136 mM NaCl, 10 mM Hepes, 2.7 mM KCl, 2 mM MgCl2, 0.1% bovine serum albumin and 0.1% glucose). Add 1/20 volume of ACD and gently mix.</li>
		<li>Pellet the platelets at 1,110 g without brake for 15 min.</li>
		<li>Discard the supernatant and carefully suspend the platelets as above in 1 mL platelet buffer (pH 7.5).</li>
		<li>Measure the platelet concentration in a 1/10 dilution with an automated hematology analyzer.</li>
		<li>Adjust the volume with platelet buffer (pH 7.5) to achieve a count of 2 x 107/mL.</li>
	</ol>
	</li>
	<li>Isolation of polymorphonuclear cells from human blood (adapted from Brinkmann et al.11 )
	<ol>
		<li>Draw 24 mL of blood in heparin (10 U/mL) as anticoagulant.</li>
		<li>Add 6 mL of 1.077 g/mL density polysucrose-sodium diatrizoate medium (see Table of Materials) to a 15-mL conical tube, and carefully layer 5-6 mL whole blood on top.</li>
		<li>Centrifuge for 20 min at 800 x g without brake.</li>
		<li>Aspirate and discard the clear yellowish top layer and transfer the lower reddish phase containing granulocytes into fresh 15-mL conical tubes. Wash cells by adding PBS containing 2 mM EDTA to a final volume of 14 mL, and centrifuge for 10 min at 300 x g without brake.</li>
		<li>In the meantime, prepare a density gradient medium (e.g., percoll, see Table of Materials) working solution by mixing 18 mL density gradient medium with 2 mL 10x PBS stock solution. Dilute this working solution with PBS (1x) to obtain 4.25 mL each of 85%, 80%, 75%, 70%, and 65% gradient medium solutions.</li>
		<li>Prepare 2 conical tubes with the density gradient by layering 2 mL of every medium percentage on top of each other. Start with the highest concentration and continue in decreasing order. Mark the layers on the tube with a water-proof marker.</li>
		<li>Carefully layer 2 mL of the cell suspensions onto each of the two prepared gradients.</li>
		<li>Centrifuge for 20 min at 800 x g without brake in a carefully balanced centrifuge.</li>
		<li>After centrifugation, remove the top layer and most of the 65% layer (first ring) with PBMCs, and collect into new tubes the white remaining interphases until the 85% layer (second ring, PMN).</li>
		<li>Wash cells by filling up the tubes with PBS 2 mM EDTA, and centrifuge for 10 min at 300 x g without brake.</li>
		<li>Remove the supernatant and suspend the sedimented cells (typically &#8805;95% are PMN) in 1 mL of Hank&#39;s buffer (pH 7.45), containing 10 mM Hepes and 0.2% human albumin (Assay buffer).</li>
		<li>Count the cells using a hemocytometer and suspend cells at 1 x 106/mL.</li>
		<li>Continue with fluorescent labeling and perfusion of leukocytes (step 3.3).</li>
	</ol>
	</li>
	<li>Preparation of adherent platelet- and vascular cell-monolayers
	<ol>
		<li>Cell culture dish preparation (cultured cells)
		<ol>
			<li>Pre-coat the 35 mm cell culture dishes with 1 mL of diluted rat tail collagen (30 &#181;g/mL diluted in PBS filtered over a 0.2 &#181;m filter).</li>
			<li>Incubate for 30 min at 37 &#176;C, discard the collagen solution, and wash the dish once with PBS.</li>
		</ol>
		</li>
		<li>Culture of vascular cells in culture dishes
		<ol>
			<li>Detach primary (e.g., HUVEC, HAoEC, HAoSMC) or immortalized vascular cells (e.g., EA.Hy926, SVEC) grown to confluence in a T75 flask with 1.5 mL cell detachment solution (e.g., accutase). Add 4.5 mL of appropriate growth medium to neutralize the cell detachment solution and determine the cell concentration with a cell counting chamber. Suspend the cells at 0.5 x 105 cells/mL in appropriate growth medium (e.g., complete endothelial cell growth medium).</li>
			<li>Add 2 mL of cell suspension to each 35 mm dish. Culture the cells in a humidified incubator at 37 &#176;C with 5% CO2 until confluence (taking 24&#8211;48 h, depending on the cell type).</li>
		</ol>
		</li>
		<li>Glass coverslip preparation (adherent platelets)
		<ol>
			<li>Immerse the glass coverslip once in HCl-EtOH (1.2 M / 50% v/v).</li>
			<li>Rinse the coverslip 2 times in ultrapure water.</li>
			<li>Dry the coverslip under N2.</li>
			<li>Pre-coat the glass coverslip with 100 &#181;L, in accordance with the position of the channel of the perfusion chamber of diluted rat tail collagen type I (30 &#181;g/mL diluted in PBS filtered over a 0.2 &#181;m filter).</li>
			<li>Incubate for 30 min at RT, discard the collagen solution, and wash the coverslip 3x with PBS.</li>
			<li>Block the collagen coated coverslip with 1% BSA in HEPES for 0.5 h at RT.</li>
			<li>Wash and dry the coverslip and mount it into the perfusion chamber. Isolate platelets from human or mouse blood as outlined in step 1.1 or 2.1, respectively.</li>
		</ol>
		</li>
		<li>Immobilization of platelets
		<ol>
			<li>Add 70 &#181;L of a 2 x 107/mL platelet suspension per channel, and incubate for 1.5 h at 37 &#176;C and 5% CO2.</li>
			<li>Carefully replace non-adherent platelets with blocking solution (5% BSA in HEPES) for at least 30 min at RT. Continue with fluorescent labeling and perfusion of leukocytes (step 3.3).</li>
		</ol>
		</li>
		<li>Stimulation of vascular cell monolayer
		<ol>
			<li>Stimulate the vascular cells by replacing the media with fresh media containing 10 ng/mL tumor necrosis factor &#945; (TNF&#945;) for 4 h at 37 &#176;C and 5% CO2.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
2. Flow-based Assay with Murine Cells
<ol>
	<li>Isolation of platelets from mouse blood
	<ol>
		<li>Draw blood by cardiac puncture using a 21 G needle (~1 mL) from anesthetized (ketamine 80 mg/kg and medetomidine 0.3 mg/kg) 6&#8211;8 week-old mice (C57Bl/6) in citrate (3.2 %) as anticoagulant.</li>
		<li>Add 1/6 volume of ACD.</li>
		<li>Centrifuge at 220 x g, medium brake for 5 min to obtain platelet rich plasma (PRP).</li>
		<li>Transfer the supernatant (~600 &#181;L) plus 1/3 of red blood cells to a new tube</li>
		<li>Centrifuge at 95 x g without brake for 6 min.</li>
		<li>Transfer the supernatant (PRP) and measure the platelet concentration in a 1/10 dilution in Tyrode&#39;s buffer (TH buffer: 136 mM NaCl, 5 mM Hepes, 2.7 mM KCl, 0.42 mM NaH2PO4*H2O, 2 mM MgCl2, 0.1% bovine serum albumin and 0.1% glucose) with an automated hematology analyzer.</li>
		<li>Wash the platelets by adding 1/25 volume of ACD + 0.1 U/mL apyrase, and centrifuge at 2,870 g, medium brake for 2 min.</li>
		<li>Discard the supernatant and add 600 &#181;L TH buffer, 1/10 volume of ACD, and 0.1 U/mL apyrase. Cut off the pipet tip (P 1000), making it wide bore, which aids in the prevention of platelet activation, and gently suspend the pellet.</li>
		<li>Centrifuge at 2,870 g, medium brake for 2 min</li>
		<li>Discard the supernatant and gently suspend the platelets in TH buffer.</li>
		<li>Adjust the volume with TH buffer to achieve a count of 2 x 107/mL.</li>
	</ol>
	</li>
	<li>Preparation of adherent platelet monolayers
	<ol>
		<li>Prepare glass coverslip (adherent platelets) as per 1.3.3.</li>
	</ol>
	</li>
	<li>Immobilization of platelets
	<ol>
		<li>Add 100 &#181;L of a 2 x 107/mL platelet suspension per channel, and incubate for 1.5 h at 37 &#176;C and 5% CO2.</li>
		<li>Carefully replace non-adherent platelets with blocking solution (5% BSA in HEPES) for at least 30 min at RT. Continue with fluorescent labeling and perfusion of leukocytes (step 3.3)</li>
	</ol>
	</li>
	<li>Stimulation of mouse platelet monolayer
	<ol>
		<li>Prior to perfusion of leukocytes (step 3.3), stimulate the platelets by perfusion of thrombin (0.5 nM) in HHHSA buffer for 3 min at 37 &#176;C.</li>
	</ol>
	</li>
</ol>
3. Fluorescent Labeling and Perfusion of Leukocytes (PMN or THP-1 for Human and RAW264.7 or Primary Mouse Monocytes for Murine Flow-Based Assay)
<ol>
	<li>Fluorescent labeling
	<ol>
		<li>Label the leukocytes (1 x 106/mL) with the cell-permeant green fluorescent nucleic acid stain (1 &#181;M). Incubate the cells for 30 min at 37 &#176;C.</li>
		<li>Wash the cells with PBS by centrifugation at 300 x g for 5 min, and suspend cells to a concentration of 0.5 x 106 cells/mL (5 mL per test condition, depending on flow rate) in assay buffer.</li>
	</ol>
	</li>
	<li>Flow chamber assay preparation
	<ol>
		<li>For leukocyte adhesion on a cell monolayer, discard the cell culture medium from the dish, and assemble it into the flow chamber. Connect tubing to the syringe. For leukocyte adhesion on immobilized platelets, connect the micro slide to the syringe. Pre-warm a water bath to 37 &#176;C.</li>
		<li>Take a perfusion syringe (50 mL), install syringe on the syringe holder, and set the pump on withdraw mode. Set the pump volume to 0 mL, and set the diameter to 26.70 mm for the 50 mL syringe.</li>
		<li>Connect an elbow luer connector to one end of the tubing. Place a luer lock coupler to the syringe and connect the tubing with the elbow luer connector to the luer lock coupler. Connect the free tubing end to the flow chamber.</li>
	</ol>
	</li>
	<li>Leukocyte perfusion and adhesion
	<ol>
		<li>For leukocyte adhesion on a cell monolayer, discard the cell culture medium from the dish, and assemble it into the flow chamber. Connect tubing to the syringe. For leukocyte adhesion on immobilized platelets, connect the micro slide to the syringe.</li>
		<li>For leukocyte perfusion and adhesion over a vascular- or platelet monolayer, place the second tubing into a 50 mL conical tube containing assay buffer and fill tubing with 1 mL pipet, then squeeze the tubing and connect it to a flow chamber.</li>
		<li>Prior to leukocyte perfusion, add 3 mM CaCl2 and 2 mM MgCl2 to the cell suspension and incubate for 5 min at 37 &#176;C.</li>
		<li>Perfuse assay buffer prior to perfusion of cells to remove possible air from the chamber and tubing.</li>
		<li>Close tube ends by squeezing them tight and switch tubing from assay buffer to cell suspension, preventing air bubbles from being trapped. Perfuse cells with appropriate flow rate/shear stress until the first cells arrive.</li>
		<li>Perfuse cells with appropriate flow rate/shear stress for 2 min, and capture at least 6 pictures between 2&#8211;6 min of rolling and adherent cells with an inverted phase contrast/fluorescence microscope (e.g., EVOS-FL) using 100X magnification connected to a digital CCD or CMOS camera. 
		NOTE: Flow rate/shear stress depends on flow chamber dimensions and viscosity of perfusate. For perfusion chamber used here (see Table of Materials): 
		&#964;=&#951;*97.1*&#934; 
		&#964;: shear stress (1 dyn/cm2) 
		&#951;: viscosity (0.015 dyn*s/cm2) 
		&#934;: Flow rate (0.67 mL/min)</li>
	</ol>
	</li>
	<li>Leukocyte de-adhesion
	<ol>
		<li>For de-adhesion, switch tubing from cell suspension to assay buffer by squeezing the tubing, preventing air bubbles from becoming trapped.</li>
		<li>Detach cells by perfusion with assay buffer with an appropriate flow rate/shear stress (See 3.3.7 Note).</li>
	</ol>
	</li>
	<li>Leukocyte transmigration
	<ol>
		<li>For leukocyte transmigration, perfuse leukocytes (step 3) until an adequate quantity of leukocytes adhere (~50 cells/view field).</li>
		<li>Replace leukocyte suspension with assay buffer.</li>
		<li>Record transmigrating cells by time lapse every 10&#8211;15 s for 30&#8211;60 min at 37 &#176;C.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

This in vitro assay is a straightforward method to investigate underlying mechanisms of leukocyte recruitment during vascular inflammation, but there are some critical points to be noted. The first requirement for successfully performing this assay is the perfusion of leukocytes over an intact and confluent vascular or platelet monolayer. This can be achieved by prior coating of the surfaces with collagen type I. In general, when working with primary vascular cells, it is important to gently detach cells using t...

Upon injury, inflammation, or infection, leukocytes quickly respond to pathogen- or damage-associated molecular patterns (PAMPs, DAMPs), change into an activated state, and move out of the blood stream to sites of inflammation and tissue damage. The ability of leukocytes to interact with their cellular and molecular environment is essential for their correct function as immune cells, as highlighted by genetic disorders such as leukocyte adhesion deficiency1. Leukocyte adhesion has been the subject of intense investigation during the past decades and this has resulted in the concept of the leukocyte adhesion cascade in the early 1990s2,3. Leukocyte adhesion is initiated by the selectin-mediated capture of leukocytes to the endothelium, causing the cells to roll over the vascular surface. This rolling enables leukocytes to scan for endothelium-bound migratory cues, e.g., chemokines, which induce the activation of integrins. Subsequently, the activated integrins mediate the binding to endothelial ligands, resulting in firm leukocyte arrest. Leukocytes may subsequently prepare to extravasate by crawling and spreading, before penetrating the endothelial monolayer and transmigrating into the underlying tissue. The basic concept of the canonical leukocyte cascade has remained largely unchanged since its introduction, with some intermediate steps added4. Nevertheless, the exact molecular mechanisms and the roles of the many players involved in leukocyte recruitment have not been clarified thus far, and leukocyte recruitment remains an important subject in the field of infection, inflammation, and (auto-) immune research.
For example, during vascular inflammatory diseases such as atherosclerosis, increased leukocyte recruitment into the vessel wall drives plaque development. Unstable atherosclerotic plaques might rupture, leading to massive activation of platelets and the coagulation system, and subsequently to occlusion of the vessel5. This may result in severe cardiovascular outcomes such as myocardial infarction or stroke. In addition, endothelial denudation as it occurs clinically, e.g. after stenting of a coronary artery, leads to a multitude of interactions of leukocytes and platelets to the exposed vessel wall interior (e.g., matrix components and smooth muscle cells) and of leukocytes with platelets covering the vascular injury. These interactions are important for the further development of the disease as monocyte-platelet interactions might drive neointima formation6,7. In addition, platelet-leukocyte interactions mediated by leukocyte integrin Mac-1 (&#945;M&#946;2) and platelet GPIb&#945;&#160;have recently been identified as novel drivers of thrombosis in mice8.
Given the wide availability of human and animal blood as a source of leukocytes and platelets for research, and the broad spectrum of isolated matrix molecules and immortalized cell lines of leukocyte and vascular origin, it is feasible to simulate leukocyte interactions under flow in a laboratory setting, using specially designed flow perfusion chambers. Many variants have been designed over the past decades, ranging from vacuum-driven to self-adhesive perfusion chambers. All variants have in common that the immobile part (e.g., cultured vascular cells or matrix proteins) is assembled into a larger leak-proof chamber equipped with a pre-defined channel enabling perfusion of fluids over the immobile part. In addition, advances in molding technology enabled the development of custom-made solutions based on silica polymers9. The viscosity and flow rate of the perfused fluids and the height of the channel mainly determine the shear stress characteristics of the flow perfusion device10. In this article, we present an in vitro method to study underlying mechanisms of the adhesion, de-adhesion, and transmigration of leukocytes under venous and arterial flow regimes. The advantage of the methods presented here is that they can be performed using a common camera-connected fluorescence microscope, and do not require a experimenters to possess high technical proficiency. The in vitro assay can be manipulated in many ways (e.g., by adding inhibitors or blocking antibodies), and is thus applicable in different models of vascular inflammation and allows the investigation of adhesion protein functions or the evaluation of specific compounds.

<table><tbody><tr><td>Inverted fluorescence microscope e.g. EVOS-FL</td><td>Life Technologies Europe bv</td><td></td><td></td></tr><tr><td>Pump &lt;em&gt;e.g.&lt;/em&gt; model: 210-CE</td><td>&amp;nbsp;world precision instruments</td><td>78-9210W</td><td></td></tr><tr><td>Falcon 35 mm TC-Treated Easy-Grip Style Cell Culture Dish</td><td>corning</td><td>353001</td><td></td></tr><tr><td>50 mL syringe</td><td>Becton Dickinson</td><td>300137</td><td></td></tr><tr><td>Silicone tubing&amp;nbsp;</td><td>VWR</td><td>228-0700</td><td></td></tr><tr><td>Elbow Luer Connector Male</td><td>Ibidi</td><td>10802</td><td></td></tr><tr><td>Female Luer Lock Coupler</td><td>Ibidi</td><td>10823</td><td></td></tr><tr><td>Flow chamber</td><td>University Hospital RWTH Aachen</td><td></td><td>Patent DE10328277A1: Baltus T, Dautzenberg R, Weber CPD. Str&amp;ouml;mungskammer zur in-vitro-Untersuchung von physiologischen Vorg&amp;auml;ngen an Zelloberfl&amp;auml;chen. 2005.&amp;nbsp;</td></tr><tr><td>Ibidi sticky slide VI 0.4</td><td>Ibidi</td><td>80608</td><td></td></tr><tr><td>Coverslips for sticky slides&amp;nbsp;</td><td>Ibidi</td><td>10812</td><td></td></tr><tr><td>Collagen Type I, rat tail</td><td>Life Technologies Europe bv</td><td>A1048301</td><td></td></tr><tr><td>Recombinant Human TNF-&amp;alpha;</td><td>Peprotech</td><td>300-01A</td><td></td></tr><tr><td>Thrombin Receptor Activator for Peptide 6 (TRAP - 6)</td><td>Anaspec / Eurogentec</td><td>24191</td><td></td></tr><tr><td>SYTO 13 green fluorescent nucleic acid stain</td><td>Life Technologies Europe bv</td><td>S7575</td><td></td></tr><tr><td>Hanks&amp;rsquo; Balanced Salt Solution 10x</td><td>Sigma-Aldrich Chemie</td><td>H4641</td><td></td></tr><tr><td>HEPES buffer solution 1M, liquid</td><td>Life Technologies Europe bv</td><td>15630049</td><td></td></tr><tr><td>BUMINATE Albumin, 25%&amp;nbsp;</td><td>Baxter</td><td>60010</td><td></td></tr><tr><td>Water for injection</td><td>B. Braun</td><td>3624315</td><td></td></tr><tr><td>Calcium chloride dihydrate</td><td>Merck</td><td>102382&amp;nbsp;</td><td></td></tr><tr><td>Magnesium chloride hexahydrate&amp;nbsp;</td><td>Sigma-Aldrich Chemie</td><td>M2670</td><td></td></tr><tr><td>Apyrase</td><td>Sigma-Aldrich Chemie</td><td>A6535</td><td></td></tr><tr><td>Primary Human Umbilical Vein Endothelial Cells (HUVEC)</td><td>Promocell GmbH</td><td>C-12203</td><td></td></tr><tr><td>Endothelial Cell Growth Medium (Ready-to-use)</td><td>Promocell GmbH</td><td>C-22010</td><td></td></tr><tr><td>Primary Human Aortic Endothelial Cells (HAoEC)</td><td>ATCC</td><td>&amp;nbsp;PCS-100-011</td><td></td></tr><tr><td>Vascular Cell Basal Medium&amp;nbsp;</td><td>ATCC</td><td>PCS-100-030</td><td></td></tr><tr><td>Endothelial Cell Growth Kit-BBE</td><td>ATCC</td><td>PCS-100-040</td><td></td></tr><tr><td>Primary Human Aortic Smooth Muscle Cells (HAoSMC)</td><td>ATCC</td><td>PCS-100-012</td><td></td></tr><tr><td>Vascular Smooth Muscle Cell Growth Kit</td><td>ATCC</td><td>PCS-100-042</td><td></td></tr><tr><td>Human endothelial cell line, EA.hy926</td><td>ATCC</td><td>CRL-2922</td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle&#039;s Medium (DMEM)</td><td>ATCC</td><td>30-2002</td><td></td></tr><tr><td>Mouse endothelial cell line, SV 40 transformed (SVEC4-10)</td><td>ATCC</td><td>CRL-2181</td><td></td></tr><tr><td>Human Monocytic cell line, THP-1</td><td>DSMZ</td><td>ACC-16</td><td></td></tr><tr><td>RPMI 1640 with Ultra-Glutamine</td><td>Lonza</td><td>BE12-702F/U1</td><td></td></tr><tr><td>Mouse monocyte/macrophage RAW264.7</td><td>ATCC</td><td>TIB-71</td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle&#039;s Medium (DMEM), high glucose</td><td>Gibco</td><td>11965092</td><td></td></tr><tr><td>Accutase</td><td>Promocell GmbH</td><td>C-41310</td><td></td></tr><tr><td>Percoll</td><td>Sigma-Aldrich Chemie</td><td>P1644</td><td></td></tr><tr><td>Histopaque 1119</td><td>Sigma-Aldrich Chemie</td><td>11191</td><td></td></tr><tr><td>10x PBS</td><td>Life Technologies Europe bv</td><td>14200075</td><td></td></tr><tr><td>BD&amp;nbsp;Vacutainer Plastic Blood Collection Tubes with Sodium Heparin</td><td>Becton Dickinson</td><td>367876</td><td></td></tr><tr><td>VACUETTE TUBE 9 ml 9NC Coagulation sodium citrate 3.2%&amp;nbsp;</td><td>Greiner Bio</td><td>455322</td><td></td></tr><tr><td>HCl/EtOH mixture (1.2 mol/L HCl and 50% ethanol) in water</td><td>Sigma-Aldrich Chemie</td><td>Prepare by mixing 0.3L HCl (4 mol/L) with 0.7L of 70% v/v ethanol in a fume hood</td><td></td></tr></tbody></table>

laminar flow-based assays to investigate leukocyte recruitment on cultured vascular cells and adherent platelets

The recruitment of leukocytes upon injury or inflammation to sites of injury or tissue damage has been investigated during recent decades and has resulted in the concept of the leukocyte adhesion cascade. However, the exact molecular mechanisms involved in leukocyte recruitment have not yet been fully identified. Since leukocyte recruitment remains an important subject in the field of infection, inflammation, and (auto-) immune research, we present a straightforward laminar flow-based assay to study underlying mechanisms of the adhesion, de-adhesion, and transmigration of leukocytes under venous and arterial flow regimes. The in vitro assay can be used to study the molecular mechanisms that underlie the interactions between leukocytes and their cellular partners in different models of vascular inflammation. This protocol describes a laminar flow-based assay using a parallel-flow chamber and an inverted phase contrast microscope connected to a camera to study the interactions of leukocytes and endothelial cells or platelets, which can be visualized and recorded then analyzed offline. Endothelial cells, platelets, or leukocytes can be pretreated with inhibitors or antibodies to determine the role of specific molecules during this process. Shear conditions, i.e. arterial or venous shear stress, can be easily adapted by the viscosity and flow rate of the perfused fluids and the height of the channel.

To study endothelial-leukocyte adhesion, fluorescently labeled THP-1 cells were perfused over a TNF&#945;- or non-stimulated endothelial monolayer for 2 min at 3 dyne/cm2. The total number of adherent monocytic cells was determined after 2 min of perfusion by capturing 6 independent fields over a period of 2-6 min. The adherent cells were quantified in at least 6 pictures captured with an inverted phase contrast/fluorescence microscope (e.g., EVOS-FL) using 10X magnifi...

Watch this Scientific Journal Video about Laminar Flow-based Assays to Investigate Leukocyte Recruitment on Cultured Vascular Cells and Adherent Platelets at JoVE.com

Laminar Flow-based Assays to Investigate Leukocyte Recruitment on Cultured Vascular Cells and Adherent Platelets

Leukocytes avidly interact with vascular cells and platelets after vessel wall injury or during inflammation. Here, we describe a straightforward laminar flow-based assay to characterize the molecular mechanisms that underlie the interactions between leukocytes and their cellular partners.

The recruitment of leukocytes upon injury or inflammation to sites of injury or tissue damage has been investigated during recent decades and has resulted ...

laminar-flow-based-assays-to-investigate-leukocyte-recruitment-on

Research

JoVE Journal

Immunology and Infection

7.5K Views.  Maastricht University. Leukocyten interactie gretig met vasculaire cellen en trombocyten na vaartuig muur letsel of tijdens ontsteking. Hier beschrijven we een eenvoudig laminaire flow gebaseerde assay karakteriseren de moleculaire mechanismen die ten grondslag liggen aan de interacties tussen leukocyten en hun cellulaire partners.

Video: Laminar Flow-based Assays to Investigate Leukocyte Recruitment on Cultured Vascular Cells and Adherent Platelets

Parallel-plate Flow Chamber and Continuous Flow Circuit to Evaluate Endothelial Progenitor Cells under Laminar Flow Shear Stress

The overall goal of this method is to describe a technique to subject adherent cells to laminar flow conditions and evaluate their response to well quantifiable fluid shear stresses1.
Our flow chamber design and flow circuit (Fig. 1) contains a transparent viewing region that enables testing of cell adhesion and imaging of cell morphology immediately before flow (Fig. 11A, B), at various time points during flow (Fig. 11C), and after flow (Fig. 11D). These experiments are illustrated with human umbilical cord blood-derived endothelial progenitor cells (EPCs) and porcine EPCs2,3.
This method is also applicable to other adherent cell types, e.g. smooth muscle cells (SMCs) or fibroblasts.
The chamber and all parts of the circuit are easily sterilized with steam autoclaving. In contrast to other chambers, e.g. microfluidic chambers, large numbers of cells (&gt; 1 million depending on cell size) can be recovered after the flow experiment under sterile conditions for cell culture or other experiments, e.g. DNA or RNA extraction, or immunohistochemistry (Fig. 11E), or scanning electron microscopy5. The shear stress can be adjusted by varying the flow rate of the perfusate, the fluid viscosity, or the channel height and width. The latter can reduce fluid volume or cell needs while ensuring that one-dimensional flow is maintained. It is not necessary to measure chamber height between experiments, since the chamber height does not depend on the use of gaskets, which greatly increases the ease of multiple experiments. Furthermore, the circuit design easily enables the collection of perfusate samples for analysis and/or quantification of metabolites secreted by cells under fluid shear stress exposure, e.g. nitric oxide (Fig. 12)6.

We are describing a method to subject adherent cells to laminar flow shear stress in a sterile continuous flow circuit. The cells' adhesion, morphology can be studied through the transparent chamber, samples obtained from the circuit for metabolite analysis and cells harvested after shear exposure for future experiments or culture.

The overall goal of this method is to describe a technique to subject adherent cells to laminar flow conditions and evaluate their response to well ...

Bioengineering

Analysis of Physiologic E-Selectin-Mediated Leukocyte Rolling on Microvascular Endothelium

E-selectin is a type-1 membrane protein on microvascular endothelial cells that helps initiate recruitment of circulating leukocytes to cutaneous, bone and inflamed tissues.  E-selectin expression is constitutive on dermal and bone microvessels and is inducible by pro-inflammatory cytokines, such as IL-1&#945;/  and TNF-&#945;, on microvessels in inflamed tissues.  This lectin receptor mediates weak binding interactions with carbohydrate counter-receptor ligands on circulating leukocytes, which results in a characteristic  rolling  behavior.  Because these interactions precede more stable adhesive events and diapedesis activity, characterization of leukocyte rolling activity and identification of leukocyte E-selectin ligands have been major goals in studies of leukocyte trafficking and inflammation and in the development of anti-inflammatory therapeutics (1-5).  The intent of this report is to provide a visual, comprehensive description of the most widely-used technology for studying E-selectin   E-selectin ligand interactions under physiologic blood flow conditions.  Our laboratory in conjunction with the Harvard Skin Disease Research Center uses a state-of-the-art parallel-plate flow chamber apparatus accompanied by digital visualization and new recording software, NIS-Elements.  This technology allows us to analyze adhesion events in real time for onscreen visualization as well as record rolling activity in a video format.  Cell adhesion parameters, such as rolling frequency, shear resistance and binding/tethering efficiency, are calculated with NIS-Elements software, exported to an Excel spreadsheet and subjected to statistical analysis.  In the demonstration presented here, we employed the parallel-plate flow chamber to investigate E-selectin-dependent leukocyte rolling activity on live human bone marrow endothelial cells (hBMEC).  Human hematopoietic progenitor KG1a cells, which express a high level of E-selectin ligand, were used as our leukocyte model, while an immortalized hBMEC cell line, HBMEC-60 cells, was used as our endothelial cell model (6).  To induce and simulate native E-selectin expression in the flow chamber, HBMEC-60 cells were first activated with IL-1 .  Our video presentation showed that parallel-plate flow analysis is a suitable method for studying physiologic E-selectin-mediated leukocyte rolling activities and that functional characterization of leukocyte E-selectin ligand(s) in the flow chamber can be ascertained by implementing protease or glycosidase digestions.

This report provides a visual depiction of parallel-plate flow chamber analysis for studying leukocyte endothelial interactions under physiologic shear stress. This method is particularly useful for investigating the role of endothelial (E)-selectin and leukocyte E-selectin ligands that trigger leukocyte rolling on endothelial cell surfaces.

E-selectin is a type-1 membrane protein on microvascular endothelial cells that helps initiate recruitment of circulating leukocytes to cutaneous, bone ...

Biology

Assessing Leukocyte-endothelial Interactions Under Flow Conditions in an Ex Vivo Autoperfused Microflow Chamber Assay

Leukocyte-endothelial interactions are early and critical events in acute and chronic inflammation and can, when dysregulated, mediate tissue injury leading to permanent pathological damage. Existing conventional assays allow the analysis of leukocyte adhesion molecules only after the extraction of leukocytes from the blood. This requires the blood to undergo several steps before peripheral blood leukocytes (PBLs) can be ready for analysis, which in turn can stimulate PBLs influencing the research findings. The autoperfused micro flow chamber assay, however, allows scientists to study early leukocytes functional dysregulation using the systemic flow of a live mouse while having the freedom of manipulating a coated chamber. Through a disease model, the functional expression of leukocyte adhesion molecules can be assessed and quantified in a micro-glass chamber coated with immobilized endothelial adhesion molecules ex vivo. In this model, the blood flows between the right common carotid artery and left external jugular vein of a live mouse under anesthesia, allowing the interaction of native PBLs in the chamber. Real-time experimental analysis is achieved with the assistance of an intravital microscope as well as a Harvard Apparatus pressure device. The application of a flow regulator at the input point of the glass chamber allows comparable physiological flow conditions amongst the experiments. Leukocyte rolling velocity is the main outcome and is measured using the National Institutes of Health open-access software ImageJ. In summary, the autoperfused micro flow chamber assay provides an optimal physiological environment to study leukocytes endothelial interaction and allows researchers to draw accurate conclusions when studying inflammation.

Assessing Leukocyte-endothelial Interactions Under Flow Conditions in an Ex Vivo Autoperfused Microflow Chamber Assay

Here, we present a protocol that allows the investigator to assess leukocyte recruitment dynamics ex vivo by connecting a chamber coated with endothelial-derived adhesion molecules to the circulatory system of a mouse. This method offers significant advantages since it allows for leukocyte assessment under relative biological conditions.

Leukocyte-endothelial interactions are early and critical events in acute and chronic inflammation and can, when dysregulated, mediate tissue injury ...

A Flow Adhesion Assay to Study Leucocyte Recruitment to Human Hepatic Sinusoidal Endothelium Under Conditions of Shear Stress

Leucocyte infiltration into human liver tissue is a common process in all adult inflammatory liver diseases. Chronic infiltration can drive the development of fibrosis and progression to cirrhosis. Understanding the molecular mechanisms that mediate leucocyte recruitment to the liver could identify important therapeutic targets for liver disease. The key interaction during leucocyte recruitment is that of inflammatory cells with endothelium under conditions of shear stress. Recruitment to the liver occurs within the low shear channels of the hepatic sinusoids which are lined by hepatic sinusoidal endothelial cells (HSEC). The conditions within the hepatic sinusoids can be recapitulated by perfusing leucocytes through channels lined by human HSEC monolayers at specific flow rates. In these conditions leucocytes undergo a brief tethering step followed by activation and firm adhesion, followed by a crawling step and subsequent transmigration across the endothelial layer. Using phase contrast microscopy, each step of this &#39;adhesion cascade&#39; can be visualized and recorded followed by offline analysis. Endothelial cells or leucocytes can be pretreated with inhibitors to determine the role of specific molecules during this process.

Leucocyte recruitment to the liver occurs within the specialized channels of the hepatic sinusoids which are lined by unique hepatic sinusoidal endothelial cells. Phase contrast microscopy of leucocyte recruitment across human hepatic sinusoidal endothelium under conditions of physiological shear stress can facilitate the elucidation of the molecular mechanisms which underlie this process.

Leucocyte infiltration into human liver tissue is a common process in all adult inflammatory liver diseases. Chronic infiltration can drive the ...

Microfluidic Assay for the Assessment of Leukocyte Adhesion to Human Induced Pluripotent Stem Cell-derived Endothelial Cells (hiPSC-ECs)

Endothelial cells (ECs) are essential for the regulation of inflammatory responses by either limiting or facilitating leukocyte recruitment into affected tissues via a well-characterized cascade of pro-adhesive receptors which are upregulated on the leukocyte cell surface upon the inflammatory trigger. Inflammatory responses differ between individuals in the population and the genetic background can contribute to these differences. Human induced pluripotent stem cells (hiPSCs) have been shown to be a reliable source of ECs (hiPSC-ECs), thus representing an unlimited source of cells that capture the genetic identity and any genetic variants or mutations of the donor. hiPSC-ECs can therefore be used for modeling inflammatory responses in donor-specific cells. Inflammatory responses can be modeled by determining leukocyte adhesion to the hiPSC-ECs under physiological flow. This step-by-step protocol provides a detailed description of the experimental setup and data analysis for the assessment of inflammatory responses in hiPSC-ECs and the analysis of leukocyte adhesion under physiological flow.

Microfluidic Assay for the Assessment of Leukocyte Adhesion to Human Induced Pluripotent Stem Cell-derived Endothelial Cells hiPSC-ECs

This step-by-step protocol provides a detailed description of the experimental setup and data analysis for the assessment of inflammatory responses in hiPSC-ECs and the analysis of leukocyte adhesion under physiological flow.

Endothelial cells (ECs) are essential for the regulation of inflammatory responses by either limiting or facilitating leukocyte recruitment into affected ...

Developmental Biology

Analyzing the Effects of Stromal Cells on the Recruitment of Leukocytes from Flow

Stromal cells regulate the recruitment of circulating leukocytes during inflammation through cross-talk with neighboring endothelial cells. Here we describe two in vitro &#8220;vascular&#8221; models for studying the recruitment of circulating neutrophils from flow by inflamed endothelial cells. A major advantage of these models is the ability to analyze each step in the leukocyte adhesion cascade in order, as would occur in vivo. We also describe how both models can be adapted to study the role of stromal cells, in this case mesenchymal stem cells (MSC), in regulating leukocyte recruitment.
Primary endothelial cells were cultured alone or together with human MSC in direct contact on Ibidi microslides or on opposite sides of a Transwell filter for 24 hr. Cultures were stimulated with tumor necrosis factor alpha (TNF&#945;) for 4 hr and incorporated into a flow-based adhesion assay. A bolus of neutrophils was perfused over the endothelium for 4 min. The capture of flowing neutrophils and their interactions with the endothelium was visualized by phase-contrast microscopy.
In both models, cytokine-stimulation increased endothelial recruitment of flowing neutrophils in a dose-dependent manner. Analysis of the behavior of recruited neutrophils showed a dose-dependent decrease in rolling and a dose-dependent increase in transmigration through the endothelium. In co-culture, MSC suppressed neutrophil adhesion to TNF&#945;-stimulated endothelium.
Our flow based-adhesion models mimic the initial phases of leukocyte recruitment from the circulation. In addition to leukocytes, they can be used to examine the recruitment of other cell types, such as therapeutically administered MSC or circulating tumor cells. Our multi-layered co-culture models have shown that MSC communicate with endothelium to modify their response to pro-inflammatory cytokines, altering the recruitment of neutrophils. Further research using such models is required to fully understand how stromal cells from different tissues and conditions (inflammatory disorders or cancer) influence the recruitment of leukocytes during inflammation.

The ability of inflamed endothelium to recruit leukocytes from flow is regulated by mesenchymal stromal cells. We describe two in vitro models incorporating primary human cells that can be used to assess neutrophil recruitment from flow and examine the role that mesenchymal stromal cells play in regulating this process.

Stromal cells regulate the recruitment of circulating leukocytes during inflammation through cross-talk with neighboring endothelial cells. Here we ...

Platelet Adhesion and Aggregation Under Flow using Microfluidic Flow Cells

Platelet aggregation occurs in response to vascular injury where the extracellular matrix below the endothelium has been exposed. The platelet adhesion cascade takes place in the presence of shear flow, a factor not accounted for in conventional (static) well-plate assays. This article reports on a platelet-aggregation assay utilizing a microfluidic well-plate format to emulate physiological shear flow conditions. Extracellular proteins, collagen I or von Willebrand factor are deposited within the microfluidic channel using active perfusion with a pneumatic pump. The matrix proteins are then washed with buffer and blocked to prepare the microfluidic channel for platelet interactions. Whole blood labeled with fluorescent dye is perfused through the channel at various flow rates in order to achieve platelet activation and aggregation. Inhibitors of platelet aggregation can be added prior to the flow cell experiment to generate IC50 dose response data.

The platelet adhesion cascade takes place in the presence of shear flow, a factor not accounted for in conventional (static) well-plate assays. This article reports on a platelet-aggregation assay utilizing a microfluidic well-plate format to emulate physiological shear flow conditions.

Platelet aggregation occurs in response to vascular injury where the extracellular matrix below the endothelium has been exposed. The platelet adhesion ...

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 ...

Live-cell Imaging of Platelet Degranulation and Secretion Under Flow

Blood platelets are essential players in hemostasis, the formation of thrombi to seal vascular breaches. They are also involved in thrombosis, the formation of thrombi that occlude the vasculature and injure organs, with life-threatening consequences. This motivates scientific research on platelet function and the development of methods to track cell-biological processes as they occur under flow conditions.
A variety of flow models are available for the study of platelet adhesion and aggregation, two key phenomena in platelet biology. This work describes a method to study real-time platelet degranulation under flow during activation. The method makes use of a flow chamber coupled to a syringe-pump setup that is placed under a wide-field, inverted, LED-based fluorescence microscope. The setup described here allows for the simultaneous excitation of multiple fluorophores that are delivered by fluorescently labeled antibodies or fluorescent dyes. After live-cell imaging experiments, the cover glasses can be further processed and analyzed using static microscopy (i.e., confocal microscopy or scanning electron microscopy).

This work describes a fluorescence microscopy-based method for the study of platelet adhesion, spreading, and secretion under flow. This versatile platform enables the investigation of platelet function for mechanistic research on thrombosis and hemostasis.

Blood platelets are essential players in hemostasis, the formation of thrombi to seal vascular breaches. They are also involved in thrombosis, the ...

An In Vitro Flow Investigation of the Effect of Stromal Cells on Leukocyte Recruitment

Source: Munir, H. et al., <a href="https://app.jove.com/v/52480">Analyzing the Effects of Stromal Cells on the Recruitment of Leukocytes from Flow.</a> J. Vis. Exp.&#160;(2015)
The video demonstrates a method for investigating the impact of stromal cells on leukocyte recruitment. In a parallel plate flow chamber model, pro-inflammatory cytokines induce the expression of adhesion receptors by endothelial cells, facilitating neutrophil recruitment and migration. The cross-talk between stromal cells and endothelial cells diminishes neutrophil recruitment to the endothelial cells.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Isolation and Culture of Primary ...

Laminar Flow-based Assays to Investigate Leukocyte Recruitment on Cultured Vascular Cells and Adherent Platelets

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