We thank Dr. Pina Colarusso and the Live Cell Imaging Facility for their assistance with imaging and image analysis; Ms. Lailey for her technical assistance; and unit 51 at the Foothills Hospital in Calgary, AB for providing human umbilical cords. Dr. K.D. Patel is an Alberta Innovates: Health Solutions Scientist. This work was supported by an operating grant from the Canadian Institutes for Health Research and equipment and infrastructure grants from the Canadian Foundation for Innovation and the Alberta Science and Research Authority.

1. Isolation and Maintenance of Human Vascular Endothelial Cells
<ol>
 <li>Level 2 biosafety procedures must be used when working with human blood and tissue. Using scissors cut the cord from the placenta and then closely examine the cord for blood clots and damage caused by clamping the cord during delivery. Cut out and discard these portions of the cord.</li>
 <li>Prepare fresh collagenase solution at 1 mg/ml by pouring 50 ml of warm cord buffer into a tube that contains 50 mg collagenase.</li>
 <li>Change into sterile gloves and open an autoclaved instrument tray containing sterilized blunt cannulas, cord clamps, scissors, three-way stop-cocks and gauze in a laminar flow hood.</li>
 <li>Identify the two arteries and the single vein present in the cord. The arteries are smaller and tend to be tightly constricted, whereas the vein is large and easy to cannulate. To flush the blood from the vein, gently insert a cannula with a two-way stopcock attached to it into one end of the vein and place a clamp around cannula to hold it firmly in position. Perfuse cord buffer through the vein. Repeat until flow through is clear and then clamp the end of the cord.</li>
 <li>Perfuse collagenase into the vein, close the stopcock, place the cord into a beaker containing warm cord buffer and incubate for 10 minutes. After 10 minutes, remove the cord and gently massage the cord to loosen endothelial cells from the lumen of vein. Drain the solution into the tube containing endothelial cell media (ECM). Flush with cord buffer twice to remove remaining cells. Drain the solution into the same tube.</li>
 <li>Centrifuge the cells at 350 x g for 10 minutes to pellet the endothelial cells.</li>
 <li>Remove the supernatant and add 10 ml of ECM to the pellet. Gently resuspend the cells in ECM, then transfer the cells to a T75 flask pre-coated with 0.2% gelatin. Pre-coating with gelatin has been done for at least 1 hour at 37 °C. Grow the cells at 37 °C and 5% CO2.</li>
 <li>The next day, gently shake the flask to dislodge any red blood cells then remove the supernatant and wash with warm Hank's balanced salt solution (HBSS). Remove HBSS and feed the cells with 10 ml of warm ECM. Examine the cells under the microscope to ensure that the red blood cells were removed. Evaluate the degree of confluence and morphology of the endothelial cells. Endothelial cells at this stage should be elongated with no sign of vacuoles.</li>
 <li>Continue to change the media every three days until cells reach confluency, then split cells using a 0.08% solution of trypsin containing 1mM EDTA. Plate endothelial cells in desired dishes pre-coated with 0.2% gelatin. Cells should reach confluence in 3-6 days.</li>
 <li>When using ibidi chambers, pre-coat each chamber with 0.2% gelatin. Remove excess gelatin and fibronectin and then add 1.5 X 106/mL of endothelial cells suspended in ECM into the chamber. Change the media every other day until confluent. Cells should be confluent in 2-3 days depending on the cord.</li>
</ol>
2. Preparation of Neutrophils
<ol>
 <li>Neutrophils were isolated from the peripheral blood of healthy human volunteers using density centrifugation. A detailed protocol with a visual demonstration for neutrophil isolation can be found in the earlier issue of this journal 6. Once isolated, resuspend neutrophils at a concentration of 1x106/mL in HBSS containing 0.5% human albumin.</li>
</ol>
3. Setting up the Ibidi Chamber
<ol>
 <li>Grow endothelial cells in a gelatin-coated ibidi chamber. Examine them every day using phase contrast microscopy until they become tightly confluent.</li>
 <li>Stimulate the endothelial cells to increase the expression of adhesion and activation molecules. For the purposes of this protocol, endothelial cells were stimulated with 10 ng/mL of recombinant TNF-α for four hours at 37 °C and 5% CO2 prior to setting up the ibidi chamber for imaging under flow conditions.</li>
 <li>Prepare the microscope, and syringe pump as visually described by Wiese and colleagues 11 using an empty ibidi chamber.</li>
 <li>Key differences between this protocol and the Wiese procedure include pre-filling the tubing to prevent exposure of the endothelial cells to air, maintaining the buffer at 37 °C using a water bath, using PharMed tubing to keep the buffer from losing temperature and using different objectives on the microscope depending on whether imaging with phase contrast or fluorescence.</li>
 <li>Once the microscope is set up and the tubing is filled with HBSS, close all the stopcocks and remove the empty ibidi chamber.</li>
 <li>Connect the ibidi chamber containing TNF-α stimulated endothelial cells to the tubing.</li>
</ol>
4. Neutrophil Recruitment and Transmigration
<ol>
 <li>Visualize the endothelial cell monolayer in the microscope using a 10x phase contrast objective.</li>
 <li>Set the syringe pump to withdraw and begin the flow of HBSS at the desired shear stress (typically between 0.5 to 2 dyn/cm2).</li>
 <li>On the inlet side, switch from HBSS to isolated neutrophils (1x106/mL) by turning the three-way stop-cock.</li>
 <li>Begin recording using a CCD camera connected either to a DVD recorder or connected directly to the computer. Start the timer when neutrophils enter the chamber. Perfuse neutrophils for four minutes. A total of 3 mL of the neutrophil suspension is sufficient for a single experiment.</li>
 <li>After four minutes, switch back to HBSS to prevent the attachment of new neutrophils. If data for total interactions, rolling and firm adhesion are desired, image 6 random fields for 10 seconds each using a 10x phase contrast objective between minutes 4 and 5.</li>
 <li>Switch to a 40x objective and record a single field of view between 5 and 10 minutes. At 10 minutes, collect between 5 and 10 random fields of view.</li>
 <li>Stop the flow and move to next chamber.</li>
</ol>
5. Fluorescent Imaging Under Flow Conditions Using an Ibidi Chamber
<ol>
 <li>Label either endothelial cells or neutrophils with a fluorescent probe of interest. For example, an anti-VE-cadherin antibody conjugated to Alexa-[547] can be used to visualize endothelial cell junctions.</li>
 <li>Assemble ibidi chamber on a microscope with differential interference contrast (DIC) and fluorescent capabilities. We use a FluoView 1000 confocal (Olympus) with an environmental chamber. Focus using an objective designed for DIC and fluorescent work.</li>
 <li>Acquire simultaneous DIC and fluorescent images using software supplied by vendor while following the time course described in section 4.</li>
</ol>
6. Analysis of Neutrophil Recruitment
<ol>
 <li>Analysis of rolling and interacting cells is described by Wiese et al11.</li>
 <li>To measure the percentage of neutrophils rolling on the endothelial monolayer count the numbers of neutrophils that have moved more than one cell diameter in a period of 5 seconds. Divide this number by the total numbers of leukocytes in the field to determine the percentage of rolling cells. By extension, the remaining cells are considered firmly adherent.</li>
 <li>Measure the rolling velocity of neutrophil by calculating the distance the neutrophil travelled in a particular time period and then dividing it by that time in seconds.</li>
 <li>Transmigration is determined by counting the number of neutrophils that have changed shape and migrated beneath the monolayer10. These neutrophils are identified by the fact that they change from being phase bright when on top of the monolayer to being phase dark when underneath the monolayer10. The number of cells transmigrated can be expressed as a percentage of the total cells in the field of view or as a raw number of transmigrated cells per unit area. In this model system, typically 50% of neutrophils transmigrate after 10 minutes.</li>
</ol>
7. Representative Results
Unstimulated endothelial cells do not support neutrophil recruitment. In contrast, stimulating endothelial cells with TNF-α results in neutrophil rolling, firm adhesion and transmigration. An example of these data is shown in Figures 1 and 2. Figure 1A shows neutrophils interacting with TNF-α-stimulated endothelial cells. These interactions can be quantified, revealing the total number of neutrophils interacting with the endothelial cells as well as the number of neutrophils rolling, firmly adherent or transmigrated (Figure 2). Neutrophil transmigration can occur at cell junctions or through the endothelial cells themselves. To differentiate between these two conditions, endothelial cells are labeled with an anti-VE-cadherin antibody and transmigration is scored as occurring paracellularly or transcellularly (Figure 1B and C). In this model, virtually all transmigration is paracellular7.
<img alt="Microscope images showing cell boundaries and structures with fluorescence visualization, cellular study." src="/files/ftp_upload/4032/4032fig1.jpg" data-altupdatedat="1761809329000" data-alt="Figure 1"> 
Figure 1. Simultaneous DIC and fluorescence imaging under flow conditions using an ibidi chamber. (A) DIC image freshly isolated human neutrophils migrating across human umbilical vein endothelial cells. The yellow arrowhead shows an adherent neutrophil; the white arrowhead shows a transmigrating neutrophil. (B) Endothelial cell junctions were stained with an anti-VE-cadherin antibody conjugated to Alexa-[547] and imaged. (C) Shows overlay of channel A and B revealing that virtually all transmigrating in this model is paracellular. <a href="https://www.jove.com/files/ftp_upload/4032/4032fig1large.jpg" target="_blank"> Click here to view larger figure</a>.
<img alt="Bar graphs of cell interactions, neutrophil adhesion, and transmigration under TNF stimulation." src="/files/ftp_upload/4032/4032fig2.jpg" data-altupdatedat="1761809329000" data-alt="Figure 2"> 
Figure 2. Endothelial cells were left unstimulated or were stimulated with 10 ng/mL of TNF-α. After 4 hours, a parallel plate flow chamber was assembled and neutrophils were perfused across at 1 dyn/cm2. (A) Neutrophil interactions were examined and measured using ImageJ software from NIH. The total interacting cells were characterized as (B) rolling, firmly adherent or (C) transmigrating. Data represent the mean + SEM of between 3 and 5 experiments. The difference between control and TNF in panels A and C was significant (p&lt;0.001).

<ol><li>Nathan, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neutrophils+and+immunity%3A+challenges+and+opportunities%5BTitle%5D)+AND+%22Nat.+Rev.+Immunol%22%5BJournal%5D)">Neutrophils and immunity: challenges and opportunities.</a> Nat. Rev. Immunol. 6, 173-182 (2006).</li>
<li>Diacovo, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neutrophil+rolling%2C+arrest%2C+and+transmigration+across+activated%2C+surface-adherent+platelets+via+sequential+action+of+P-selectin+and+the+beta+2-integrin+CD11b%2FCD18%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">Neutrophil rolling, arrest, and transmigration across activated, surface-adherent platelets via sequential action of P-selectin and the beta 2-integrin CD11b/CD18.</a> Blood. 88, 146-157 (1996).</li>
<li>Ley, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Getting+to+the+site+of+inflammation%3A+the+leukocyte+adhesion+cascade+updated%5BTitle%5D)+AND+%22Nat.+Rev.+Immunol%22%5BJournal%5D)">Getting to the site of inflammation: the leukocyte adhesion cascade updated.</a> Nat. Rev. Immunol. 7, 678-689 (2007).</li>
<li>Petri, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Endothelial+LSP1+is+involved+in+endothelial+dome+formation%2C+minimizing+vascular+permeability+changes+during+neutrophil+transmigration+in+vivo%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">Endothelial LSP1 is involved in endothelial dome formation, minimizing vascular permeability changes during neutrophil transmigration in vivo.</a> Blood. 117, 942-952 (2011).</li>
<li>Chavakis, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+junctional+adhesion+molecule-C+promotes+neutrophil+transendothelial+migration+in+vitro+and+in+vivo%5BTitle%5D)+AND+%22J.+Biol.+Chem%22%5BJournal%5D)">The junctional adhesion molecule-C promotes neutrophil transendothelial migration in vitro and in vivo.</a> J. Biol. Chem. 279, 55602-55608 (2004).</li>
<li>Oh, H., Siano, B., Diamond, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neutrophil+Isolation+Protocol%5BTitle%5D)+AND+%22J.+Vis.+Exp%22%5BJournal%5D)">Neutrophil Isolation Protocol.</a> J. Vis. Exp. (17), e745(2008).</li>
<li>Liu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Regulation+of+leukocyte+transmigration%3A+cell+surface+interactions+and+signaling+events%5BTitle%5D)+AND+%22J.+Immunol%22%5BJournal%5D)">Regulation of leukocyte transmigration: cell surface interactions and signaling events.</a> J. Immunol. 172, 7-13 (2004).</li>
<li>Alcaide, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neutrophil+recruitment+under+shear+flow%3A+it%27s+all+about+endothelial+cell+rings+and+gaps%5BTitle%5D)+AND+%22Microcirculation%22%5BJournal%5D)">Neutrophil recruitment under shear flow: it's all about endothelial cell rings and gaps.</a> Microcirculation. 16, 43-57 (2009).</li>
<li>Jutila, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Measurement+of+neutrophil+adhesion+under+conditions+mimicking+blood+flow%5BTitle%5D)+AND+%22Neutrophil+Methods+and+Protocols%22%5BJournal%5D)">Measurement of neutrophil adhesion under conditions mimicking blood flow.</a> Neutrophil Methods and Protocols. 412, 239-256 (2007).</li>
<li>Cuvelier, S. L., Patel, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Studying+leukocyte+rolling+and+adhesion+in+vitro+under+flow+conditions%5BTitle%5D)+AND+%22Basic+Cell+Culture+Protocols%22%5BJournal%5D)">Studying leukocyte rolling and adhesion in vitro under flow conditions.</a> Basic Cell Culture Protocols. 290, 331-342 (2005).</li>
<li>Wiese, G., Barthel, S. R., Dimitroff, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Analysis+of+Physiologic+E-Selectin-Mediated+Leukocyte+Rolling+on+Microvascular+Endothelium%5BTitle%5D)+AND+%22J.+Vis.+Exp%22%5BJournal%5D)">Analysis of Physiologic E-Selectin-Mediated Leukocyte Rolling on Microvascular Endothelium.</a> J. Vis. Exp. (24), e1009(2009).</li>
<li>Petzelbauer, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Heterogeneity+of+dermal+microvascular+endothelial+cell+antigen+expression+and+cytokine+responsiveness+in+situ+and+in+cell+culture%5BTitle%5D)+AND+%22J.+Immunol%22%5BJournal%5D)">Heterogeneity of dermal microvascular endothelial cell antigen expression and cytokine responsiveness in situ and in cell culture.</a> J. Immunol. 151, 5062-5072 (1993).</li>
<li>Bonder, C. S., Kubes, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Modulating+leukocyte+recruitment+to+splanchnic+organs+to+reduce+inflammation%5BTitle%5D)+AND+%22Am+J+Phys+-+Gastrointestinal+and+Liver+Phys%22%5BJournal%5D)">Modulating leukocyte recruitment to splanchnic organs to reduce inflammation.</a> Am J Phys - Gastrointestinal and Liver Phys. 284, G729-G733 (2003).</li>
<li>Cuvelier, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Eosinophil+adhesion+under+flow+conditions+activates+mechanosensitive+signaling+pathways+in+human+endothelial+cells%5BTitle%5D)+AND+%22J.+Exp.+Med%22%5BJournal%5D)">Eosinophil adhesion under flow conditions activates mechanosensitive signaling pathways in human endothelial cells.</a> J. Exp. Med. 202, 865-876 (2005).</li>
<li>Massia, S. P., Hubbell, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Human+endothelial+cell+interactions+with+surface-coupled+adhesion+peptides+on+a+nonadhesive+glass+substrate+and+two+polymeric+biomaterials%5BTitle%5D)+AND+%22J.+Biomed.+Mat.+Res%22%5BJournal%5D)">Human endothelial cell interactions with surface-coupled adhesion peptides on a nonadhesive glass substrate and two polymeric biomaterials.</a> J. Biomed. Mat. Res. 25, 223-242 (1991).</li>
</ol>

No conflicts of interest declared.

Researchers have routinely used endothelial cells from different vascular beds to study neutrophil recruitment and transmigration. Examples include, but are not limited to, dermal microvascular endothelial cells12, liver sinusoidal endothelial cells13 and endothelial cells from human umbilical vein10. Among them HUVEC gained wide popularity because of their relative ease of isolation and availability. HUVEC are a powerful in vitro model system that mimics many endothelial process...

<table><tbody><tr><td>Collagenase Type 1</td><td>Worthington</td><td>4197</td><td></td></tr><tr><td>Cord buffer</td><td></td><td></td><td>Composition: 140 mM NaCl 4 mM KCl 10 mM D-glucose in 1mM NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;/Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt; buffer at pH 7.4</td></tr><tr><td>Endothelial Cell Media (ECM)</td><td></td><td></td><td>M199 with eagle salts supplemented with 16% human serum containing 100 units of penicillin 100 &amp;mu;g of streptomycin and 0.3 mg of L-glutamine/ml</td></tr><tr><td>M199</td><td>GIBCO</td><td>31100-035</td><td></td></tr><tr><td>Penicillin Streptomycin Glutamate (100X)</td><td>Invitrogen</td><td>10378-016</td><td></td></tr><tr><td>Ibidi chambers</td><td>Ibidi</td><td>80606</td><td></td></tr><tr><td>TNF-&amp;alpha;</td><td>Prepro Tech</td><td>300-01A</td><td></td></tr><tr><td>Human Albumin 20% solution</td><td>Gemini Bioproducts</td><td>800120050</td><td></td></tr><tr><td>HBSS without Ca&lt;sup&gt;2+&lt;/sup&gt; and Mg&lt;sup&gt;2+&lt;/sup&gt;</td><td>Sigma</td><td>H2487-10X</td><td></td></tr><tr><td>HBSS with Ca&lt;sup&gt;2+&lt;/sup&gt; and Mg&lt;sup&gt;2+&lt;/sup&gt;</td><td>Sigma</td><td>H1387-10X</td><td></td></tr></tbody></table>

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.

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, ...

The overall goal of this procedure is to isolate high quality human endothelial cells and to use them to examine the mechanisms regulating neutrophil trans migration, underflow conditions. This is accomplished by first isolating and plating human umbilical vein endothelial cells. The second step is to split the cells into IBD chambers.Next freshly isolated human neutrophils are profused across activated endothelial cells, resulting in neutrophil rolling firm adhesion and transmigration. The final step is to quantify transmigration using video microscopy. Ultimately fluorescent and brightfield microscopy can be used to evaluate neutrophil transmigration across activated endothelial cells.Endothelial cell isolation is easy to describe, but difficult to learn in part because key steps require visualization of the umbilical cord. For this reason, visual demonstration of this method is critical. This method will help us to understand key questions in the inflammation field, such as how leukocyte recruitment happens under flow condition, and how endothelial cell behaves under flow condition will be better.Understood.Though this method can provide insight into neutrophil recruitment during inflammation. It can also be applied to leukocyte rare population such as natural killer cells and eosinophil. Demonstrating this procedure along with Ritu Sharma is Hong Jang, a technician in my laboratory At least one hour before the procedure preco, and then incubate a T 75 flask with gelatin at 37 degrees Celsius.Then begin the endothelial cell isolation procedure in the laminar flow hood by using a pair of scissors to cut an umbilical cord from a placenta. After changing into sterile gloves, closely examine the cord and discard any portions that contain blood clots or are damage from cord clamping during delivery. Then identify the two arteries and the single vein present in the cord.Next, gently insert a cannula with a two-way stop cock attached to it into one end of the vein, and then place a clamp around the cannula to hold it firmly in position. One of the trickiest parts of the procedure is perfusing the umbilical cord. To ensure this is done successfully, perfuse the cord with excess buffer and watch the flow through until it runs clear.Perfuse the vein with cord buffer until the flow through is clear and then clamp the end of the cord. Occasionally during perfusion, a small hole is revealed because physicians have removed cord blood from the umbilical cord. In these situations, hemostats can be used to block the hole and perfusion can continue while isolating endothelial cells.It's critically important to precisely time the collagenase treatment. Too little time and not enough endothelial cells will be harvested too long and contaminating smooth muscle cells or damage to the endothelial cells can occur. Then perfuse the vein with freshly prepared warm collagenase.Close the stop cock and incubate the cord in a beaker containing warm cord buffer. After 10 minutes, remove and then gently massage the cord to loosen the endothelial cells from the lumen of the vein. Drain the solution into a tube containing five milliliters of endothelial cell media or ECM flushing the cord with cord buffer twice to remove any remaining cells.Now spin down the cells for 10 minutes at three 50 times G.At room temperature, remove the supernatant and then resuspend the pellet. In 10 milliliters of endothelial cell media, transfer the cells to the gelatin coated T 75 flask and culture, the endothelial cells overnight at 37 degrees Celsius and 5%CO2. The next day, gently shake the flask to dislodge any red blood cells.Then remove the supernatant and wash the endothelial cell culture with warm HBSS. After removing the HBSS feed the cells with 10 milliliters of warm ECM. Then examine the cells under the microscope to ensure that the red blood cells were removed and to evaluate the degree of confluence and morphology of the endothelial cells.Continue to change the media every three days until the cells reach co fluency. At that point, split the cells using a trypsin and EDTA solution. Then harvest the detached cells and resuspend them in ECM.Finally, coat each well of an IBD chamber with gelatin. Remove the excess gelatin and then add 1.5 times 10 to the six per milliliter of the endothelial cell suspension into each well of the chamber. Begin by inducing the upregulation of adhesion and activation molecule cell surface expression by incubating the endothelial cells with an appropriate stimulant while the endothelial cells are being activated.Isolate neutrophils from the peripheral blood of healthy human volunteers by density centrifugation as previously published, and then suspend the cells at a concentration of one times 10 to the six per milliliter in HBSS plus human albumin. After setting up the microscope, attach the IBD chamber containing the stimulated endothelial cells to the tubing of the microscope. Select the 10 x phase contrast objective, and then set the syringe pump to withdraw and begin the flow of HBSS at the desired shear stress.Briefly stop the flow by turning the three-way stop cock on the outlet side to the off position and switching the inlet line from HBSS to isolated neutrophils. Start the flow again by turning the stop cock back to the on position. Then begin recording using a CCD camera connected to A DVD recorder.Start the timer. When neutrophils enter the chamber after four minutes, switch back to HBSS to prevent the attachment of new neutrophils. If data for total interactions rolling and firm adhesion are desired, use a 10 x phase contrast.Objective to image six random fields for 10 seconds each between minutes four and five. Finally switch to a 40 x objective and record a single field of view at the time of interest. After the neutrophil perfusion at 10 minutes, collect between five and 10 random fields of view.Unstimulated endothelial cells do not support neutrophil recruitment. Therefore, the following figures show neutrophils interacting with TNF alpha stimulated endothelial cells. Note the adherent neutrophil as indicated by the yellow arrowhead and the trans migrating neutrophil as denoted by the white arrowhead.Neutrophil transmigration can occur at cell junctions or through the endothelial cells themselves. To differentiate between these two conditions, endothelial cells were labeled with an anti VE coherent antibody. As seen here in red transmigration is scored as occurring paracellular or trans cellularly.In this model, virtually all transmigration is paracellular as demonstrated by this figure showing the overlay of the previous two figures. In this first of three graphs, neutrophil interactions were examined and measured using image J software. Note that significantly more neutrophil endothelial cell interactions occurred between neutrophils and TNF alpha activated endothelial cells than with the control unstimulated endothelial cells.The total interacting cells were further characterized as rolling or firmly adherent or trans migrating as before. Significantly more neutrophil transmigration occurred in the wells containing activated endothelial cells Following isolation of the endothelial cells. The other methods like western blotting real time PCR and endothelial resistance can be performed in order to answer additional questions related to endothelial cell biology After development.This method will help the researchers in the field of inflammation and vascular biology to understand how leukocyte recruitment happens under flow condition in a human model system. After watching this video, you should have a good understanding of how to isolate human umbilical vein endothelial cells and use them to study neutrophil transmigration under flow conditions using an ity chamber.

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