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 &deg;C. Grow the cells at 37 &deg;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-&alpha; for four hours at 37 &deg;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 &deg;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-&alpha; 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-&alpha; 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-&alpha;-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="Figure 1" src="/files/ftp_upload/4032/4032fig1.jpg" /> 
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="Figure 2" src="/files/ftp_upload/4032/4032fig2.jpg" /> 
Figure 2. Endothelial cells were left unstimulated or were stimulated with 10 ng/mL of TNF-&alpha;. 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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophils+and+immunity:+challenges+and+opportunities.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+rolling,+arrest,+and+transmigration+across+activated,+surface-adherent+platelets+via+sequential+action+of+P-selectin+and+the+beta+2-integrin+CD11b/CD18.">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/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>Petri, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+LSP1+is+involved+in+endothelial+dome+formation,+minimizing+vascular+permeability+changes+during+neutrophil+transmigration+in+vivo.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+junctional+adhesion+molecule-C+promotes+neutrophil+transendothelial+migration+in+vitro+and+in+vivo.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+Isolation+Protocol.">Neutrophil Isolation Protocol.</a> J. Vis. Exp. (17), e745 (2008).</li><li>Liu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+leukocyte+transmigration:+cell+surface+interactions+and+signaling+events.">Regulation of leukocyte transmigration: cell surface interactions and signaling events.</a> J. 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(24), e1009 (2009).</li><li>Petzelbauer, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneity+of+dermal+microvascular+endothelial+cell+antigen+expression+and+cytokine+responsiveness+in+situ+and+in+cell+culture.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulating+leukocyte+recruitment+to+splanchnic+organs+to+reduce+inflammation.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eosinophil+adhesion+under+flow+conditions+activates+mechanosensitive+signaling+pathways+in+human+endothelial+cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+endothelial+cell+interactions+with+surface-coupled+adhesion+peptides+on+a+nonadhesive+glass+substrate+and+two+polymeric+biomaterials.">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 border="1">
	<tbody>
		<tr>
			<td>Reagent and Equipment</td>
			<td>Company</td>
			<td>Catalogue number </td>
			<td>Comments </td>
		</tr>
		<tr>
			<td>Collagenase Type 1</td>
			<td>Worthington</td>
			<td>4197</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cord buffer</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Composition: 140 mM NaCl 4 mM KCl 10 mM D-glucose in 1mM NaH2PO4/Na2HPO4 buffer at pH 7.4</td>
		</tr>
		<tr>
			<td>Endothelial Cell Media (ECM)</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>M199 with eagle salts supplemented with 16% human serum containing 100 units of penicillin 100 &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>&nbsp;</td>
		</tr>
		<tr>
			<td>Penicillin Streptomycin Glutamate (100X)</td>
			<td>Invitrogen</td>
			<td>10378-016</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ibidi chambers</td>
			<td>Ibidi</td>
			<td>80606</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>TNF-&alpha;</td>
			<td>Prepro Tech</td>
			<td>300-01A</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Human Albumin 20% solution</td>
			<td>Gemini Bioproducts</td>
			<td>800120050</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>HBSS without Ca2+ and Mg2+</td>
			<td>Sigma</td>
			<td>H2487-10X</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>HBSS with Ca2+ and Mg2+</td>
			<td>Sigma</td>
			<td>H1387-10X</td>
			<td>&nbsp;</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.

Watch this Scientific Journal Video about Isolation of Human Umbilical Vein Endothelial Cells and Their Use in the Study of Neutrophil Transmigration Under Flow Conditions at JoVE.com

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

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, ...

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Immunology and Infection

17.0K Views.  University of Calgary. 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.

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

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 ...

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 ...

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 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.

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 ...

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the shear stress of flowing blood.
To identify the bacterial and host factors that contribute to vascular adhesion of microorganisms, appropriate models that study these interactions under physiological shear conditions are needed. Here, we describe an in vitro flow chamber model that allows to investigate bacterial adhesion to different components of the extracellular matrix or to endothelial cells, and an intravital microscopy model that was developed to directly visualize the initial adhesion of bacteria to the splanchnic circulation in vivo. These methods can be used to identify the bacterial and host factors required for the adhesion of bacteria under flow. We illustrate the relevance of shear stress and the role of von Willebrand factor for the adhesion of Staphylococcus aureus using both the in vitro and in vivo model.

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

To study the interaction of bacteria with the blood vessels under shear stress, a flow chamber and an in vivo mesenteric intravital microscopy model are described that allow to dissect the bacterial and host factors contributing to vascular adhesion.

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the ...

The Use of Flow Cytometry to Assess the State of Chromatin in T Cells

During a proper immune response, quiescent T cells become activated upon antigen presentation to their antigen-specific T cell receptor. This leads to clonal proliferation of only those T cells that bear a receptor that recognizes the antigen. Chromatin decondensation is a hallmark of T cell activation and is required for T cells to acquire the ability to proliferate after antigen engagement. This change in chromatin condensation can be detected using antibodies raised against histone proteins. These antibodies cannot bind to their epitopes in na&#239;ve T cells as well as they can in activated T cells. We describe how to simultaneously stain T cell-specific surface markers, track viability with a fixable dead cell stain, and measure chromatin status via intracellular staining of Histone H3 proteins. Stained cells are analyzed by flow cytometry and chromatin condensation status is measured as the mean fluorescence intensity (MFI) of the Histone H3 stain. Chromatin decondensation during T cell activation is demonstrated as an increase in the MFI

Flow cytometry can be utilized to assess the state of chromatin within T cells. This protocol allows scientists to interpret evidence of chromatin decondensation during T cell activation demonstrated by an increase in the mean florescence intensity (MFI) of fluorescent Histone H3 antibodies

During a proper immune response, quiescent T cells become activated upon antigen presentation to their antigen-specific T cell receptor. This leads to ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and their important role in lung development and function, as well as their lung-localized responses to infection and inflammation. To date, no unified method for identification, isolation, and handling of alveolar macrophages from humans and mice exists. Such a method is needed for studies on these important innate immune cells in various experimental settings. The method described here, which can be easily adopted by any laboratory, is a simplified approach to harvesting alveolar macrophages from bronchoalveolar lavage fluid or from lung tissue and maintaining them in vitro. Because alveolar macrophages primarily occur as adherent cells in the alveoli, the focus of this method is on dislodging them prior to harvest and identification. The lung is a highly vascularized organ, and various cell types of myeloid and lymphoid origin inhabit, interact, and are influenced by the lung microenvironment. By using the set of surface markers described here, researchers can easily and unambiguously distinguish alveolar macrophages from other leukocytes, and purify them for downstream applications. The culture method developed herein supports both human and mouse alveolar macrophages for in vitro growth, and is compatible with cellular and molecular studies.

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

This communication describes methodologies for isolation and culture of alveolar macrophages from humans and murine models for experimental purposes.

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...

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 ...

Preparation of Whole Bone Marrow for Mass Cytometry Analysis of Neutrophil-lineage Cells

In this article, we present a protocol that is optimized to preserve neutrophil-lineage cells in fresh BM for whole BM CyTOF analysis. We utilized a myeloid-biased 39-antibody CyTOF panel to evaluate the hematopoietic system with a focus on the neutrophil-lineage cells by using this protocol. The CyTOF result was analyzed with an open-resource dimensional reduction algorithm, viSNE, and the data was presented to demonstrate the outcome of this protocol. We have discovered new neutrophil-lineage cell populations based on this protocol. This protocol of fresh whole BM preparation may be used for 1), CyTOF analysis to discover unidentified cell populations from whole BM, 2), investigating whole BM defects for patients with blood disorders such as leukemia, 3), assisting optimization of fluorescence-activated flow cytometry protocols that utilize fresh whole BM.

Here, we present a protocol to process fresh bone marrow (BM) isolated from mouse or human for high-dimensional mass cytometry (Cytometry by Time-Of-Flight, CyTOF) analysis of neutrophil-lineage cells.

In this article, we present a protocol that is optimized to preserve neutrophil-lineage cells in fresh BM for whole BM CyTOF analysis. We utilized a ...