This work was supported by the UCSF Department of Anesthesia and Perioperative Care.

1. Plating and Maintenance of Microvascular Endothelial Cells
<ol>
	<li>Thaw and grow your microvascular endothelial cells according to the manufacturers supplied instructions. In this protocol, cells were grown in EGM-2 MV media (Lonza), and it is recommended that all experiments are performed within two weeks of thawing to minimize any variation due to passage number. Our experiments with HMVEC-Lung are performed between passages 4 to 9.</li>
	<li>Day 1: When cells reach 80-90% confluency, trypsinize and resuspend at 120,000 cells per ml. Plate 36,000 cells (0.3 ml) per well into 48-well, polystyrene tissue culture plate(s). Include wells of HMVEC that will not be incubated with neutrophils in order to obtain a background fluorescence reading. Unless using 48-well plates specifically designed for fluorescence assays, alternating rows or columns will minimize any light contamination from neighboring wells. NOTE: Wells can be pre-coated with poly-lysine, collagen or fibronectin.</li>
	<li>Day 2: Add fresh media.</li>
	<li>Day 3: By phase contrast microscopy, confirm the cells are healthy (i.e. &#34;cobblestone&#34; appearance and little cell debris), and they are 100% confluent (Figure 1). If the cells are not 100% confluent, change the media every day until they are ready.</li>
	<li>Once the HMVEC monolayers are ready for treatment, wash the cells once with Hank&#39;s balanced salt solution (HBSS) and add 0.3 ml of fresh EGM-2 MV media or media containing inflammatory agonist to the appropriate wells. In this protocol the HMVEC-Lung were treated with TNF&#945; (100 ng/ml) (PeproTech, New Jersey) for 3 hr since this is a well-described activator of ECs 21. NOTE: It is recommended that you time your experiment so that your activated HMVEC cells (Step 4.1) and calcein AM labeled neutrophils (Step 3.5) are ready to use at the same time. It takes us approximately 2.5 hr to isolate and label neutrophils.</li>
</ol>
2. Neutrophil Isolation using Polymorphprep
<ol>
	<li>Isolate neutrophils as described previously 22, or with a ready-made density gradient solution to isolate polymorphonuclear granulocytes from whole blood. Employing Polymorphprep following the protocol by Nuzzi, et al. 2007 results in yields of approximately 2-3 x 106 neutrophils per ml of whole blood 23. NOTE: To avoid excessive erythrocyte lysis, a dextran sedimentation to remove red blood cells may be performed prior to density gradient centrifugation 24.</li>
	<li>Bring the Polymorphprep density gradient solution to RT.</li>
	<li>Collect 30 ml of whole blood from a healthy human volunteer in the presence of an anti-coagulant such as heparin, citrate or EDTA. The blood should be used within 2 hr, and kept between 18-22 &deg;C.</li>
	<li>Layer 5 ml of whole blood over 5 ml of the density gradient solution in a 15 ml conical tube (Figure 2A). Avoid altering volumes of blood and the density gradient solution as this may change the subsequent centrifugation conditions.</li>
	<li>Centrifuge at 450 x g for 30 min at 18-22 &deg;C. Make sure to slowly speed up and down the centrifuge (i.e. turn off the centrifuge brake), and balance the tubes well to prevent vibrations to help reduce activation of the neutrophils and prevent the mixing of layers. Longer centrifugation times or higher centrifugal force will result in the lower leukocyte layer, which contains neutrophils, to migrate further down towards to the pelleted red blood cells.</li>
	<li>Aspirate the plasma and upper leukocyte band containing PBMCs to prevent the contamination of the layer containing neutrophils (Figure 2B).</li>
	<li>Use a 1-2 ml serological pipet to remove the lower leukocyte layer containing neutrophils. Combine the neutrophil layers into 30 ml of PBS (without Ca2+ and Mg2+) in a 50 ml conical tube.</li>
	<li>Bring the volume up to 50 ml with PBS (without Ca2+ and Mg2+) and centrifuge at 450 x g for 10 min at 18-22 &deg;C.</li>
</ol>
Note: Steps 2.9 and 2.10 are optional but highly recommended.
<ol start="9">
	<li>Aspirate the supernatant. Resuspend the neutrophils in 8 ml of sterile water for 30 sec to lyse red blood cells, immediately add the cell suspension to 2 ml of 5x PBS (without Ca2+ and Mg2+) and gently mix by hand. DO NOT incubate cells in water for longer than 30 sec since significant neutrophil death can occur.</li>
	<li>Centrifuge at 250 x g for 5 min at 18-22 &deg;C.</li>
	<li>Wash the cells once with 10 ml of PBS (without Ca2+ and Mg2+) and centrifuge again at 250 x g for 5 min at 18-22 &deg;C.</li>
	<li>Resuspend the neutrophils in 10 ml of RPMI-1640 (without phenol red) and count the live cells using the trypan blue dye exclusion method. Avoid prolonged periods of cell densities greater than 5 x 106 per ml since this may result in activation of the neutrophils.</li>
</ol>
3. Neutrophil Labeling with Calcein AM
<ol>
	<li>6 x 105 neutrophils are used per well of a 48-well plate.</li>
	<li>After counting, transfer the resuspended cells to a 50 ml conical tube and dilute the neutrophils to 2-4 x 106 cells per ml with phenol red-free RPMI-1640.</li>
	<li>Add calcein AM stock solution to 3 &#956;M (i.e. 2.98 &#956;l of calcein AM stock (1 mg/ml) per ml of media) and mix well by gently flicking the tube. Greater than 5 &#956;M calcein AM will result in leakage from the neutrophils. Note: Non-fluorescent calcein AM is cleaved within the cells by endogenous esterases to produce the highly fluorescent molecule calcein (i.e. dead cells will not fluoresce).</li>
	<li>Cover the tube with aluminum foil and incubate for 30 min at 37 &#176;C in the dark. Note: Longer incubation times may result in more calcein being liberated from the neutrophils over the course of the adhesion assay.</li>
	<li>Centrifuge at 450 x g for 5 min at 18-22 &deg;C, and wash the neutrophils 2x with 10 ml of RPMI-1640 (without phenol red; pre-warmed to 37 &deg;C). After resuspending the cells for the second wash, first pass the neutrophils though a 40 &#956;M sterile filter to remove clumps and then centrifuge once more at 450 x g for 5 min at 18-22 &deg;C.</li>
	<li>Resuspend the neutrophils in RPMI 1640 (without phenol red; pre-warmed to 37 &#176;C) at 2 x 106 neutrophils per ml.</li>
</ol>
4. Neutrophil Binding Assay
<ol>
	<li>Wash the HMVEC monolayers 3x with 0.5 ml of filter-sterilized (0.22 &#956;m) RPMI 1640 (without phenol red) containing 3% BSA per well.</li>
	<li>Aspirate the media and add 6 x 105 calcein-labeled neutrophils (0.3 ml of cell suspension), or 0.3 ml of RPMI 1640 (without phenol red) without neutrophils, to the appropriate wells of the 48-well plate. Note: This is the equivalent of 8 x 105 neutrophils per cm2.</li>
	<li>Incubate for 20 min in a 37 &#176;C, 5% CO2 incubator.</li>
	<li>Total Neutrophil Fluorescence (PRE wash): Measure the fluorescence intensity of each well with an excitation wavelength of 485 nm and an emission wavelength of 520 nm (fluorescein filter set) in a FLUOstar, or equivalent, spectrophotometer. Perform this step just prior to the incubation end point. Note: In this protocol, calcein excitation and detection of the emitted light were performed from the top of the uncovered wells; however, both can also be performed from the bottom of the wells.</li>
	<li>Wash the wells containing HMVEC monolayers and neutrophils 5x with 0.5 ml per well of PBS (w/ Ca2+ and Mg2+). Remove the media and washes by inverting the plate, and patting gently onto paper towels to remove the remaining liquid.</li>
	<li>Add back 0.3 ml of RPMI 1640 (without phenol red) per well.</li>
	<li>Adherent Neutrophil Fluorescence (POST wash): Measure the fluorescence intensity of each well as in Step 4.4.</li>
	<li>Determine the percent adherence and relative adherence per well:</li>
</ol>
Note: At this stage, images of the calcein-labeled neutrophils can be obtained by using a fluorescence capable microscope equipped with standard fluorescein filters. The images in Figure 4 were obtained on an Olympus IX51 inverted microscope equipped with a Retiga 2000R camera (Q Imaging) using Q-Capture Pro7 software (Q Imaging).

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Biol. Chem. 270, 21129-21136 (1995).</li></ol>

Authors have nothing to disclose.

The most critical steps for a successful neutrophil / microvascular endothelial cell adhesion assay are: 1) Use of low passage number (&lt;9), healthy endothelial cells; 2) Maintaining the isolated neutrophils at a low density (i.e. &lt;5 x 106 cells / ml) and using them within two hours of isolation; and 3) Fastidious washing of the calcein AM-labeled neutrophils and use of the calcein AM-labeled neutrophils in a timely fashion to minimize EC contamination and loss of calcein from the neutroph...

Since the vascular endothelium is in direct contact with the circulating blood, it is uniquely situated to initiate a rapid inflammatory response during infection or injury. Endothelial cells (ECs) express pattern recognition receptors that recognize a variety of conserved bacterial components and danger molecules, and receptors for host inflammatory mediators such as TNF&alpha;. Activation of these receptors induces ECs to secrete cytokines (e.g. IL-6, IL-8, CXCL1 and CCL2), and to upregulate adhesion molecules (e.g. E/P-selectin, VCAM-1 and ICAM-1) at their cell surface 1,2. These molecules all facilitate the localization of leukocytes to sites of infection and injury in order to clear the host of the infectious agents and initiate tissue repair 3,4.The neutrophil response to infection involves a well-coordinated interplay between the vascular endothelium and the responding neutrophils. Upon EC activation, IL-8 is secreted and forms an intravascular gradient on the endothelium that allows neutrophils to home in to the site of infection or injury 5,6. E/P-selectins mediate neutrophil capture and rolling through relatively weak associations with glycomolecules on the neutrophil cell surface. These interactions, along with IL-8 binding to its cognate receptors, facilitate the robust, integrin-mediated attachment and eventual arrest of neutrophils on the endothelial cell surface 7-10. After arrest, neutrophils can migrate out of the vasculature to the specific sites of infection to directly eliminate pathogens, generate neutrophil extracellular traps to prevent the spread of pathogens, promote wound healing and release additional factors that recruit other leukocytes such as monocytes, macrophages and dendritic cells 11-17.
Described in this report is an in vitro method to quantitate neutrophil adherence to microvascular ECs after activation by the host inflammatory mediator TNF&alpha;. This assay is designed to assess the activation of ECs, and not neutrophils. Primary human neutrophils are first isolated using density gradient separation, and are then labeled with calcein acetoxymethyl (AM). Esterases within the live cells hydrolyze calcein AM to the highly fluorescent calcein molecule with an excitation of 492-495 nm and emission of 513-516 nm 18. The fluorescently-labeled neutrophils are then incubated with EC monolayers, and non-adherent neutrophils are subsequently removed. The fluorescence of the remaining, bound neutrophils is then measured using a fluorescence spectrophotometer, and calculated as a percent of total neutrophil fluorescence input per well. This method has the additional advantage that the bound calcein-labeled neutrophils used in spectrophotometry can be directly visualized using fluorescence microscopy to give a more qualitative read out of EC activation. Since this assay is performed under static conditions, only the very initial events that occur in the neutrophil adhesion cascade will be assessed. This is confirmed in this report using E-selectin blocking antibodies to show that neutrophil adherence to TNF&alpha;-treated human lung microvascular EC (HMVEC-Lung) monolayers is drastically reduced when the interaction with E-selectin is disrupted.
In addition to TNF&alpha;, we have successfully used this assay to determine the extent of human umbilical vein EC (HUVEC) activation by the Toll-like receptor 1/2 agonists peptidoglycan-associated lipoprotein (PAL), murein lipoprotein (MLP) and Pam3Cys and HMVEC-Lung activation by Pam3Cys 19,20. In addition, we successfully used this assay with kinase inhibitors and after RNAi-mediated knockdown of surface and cytoplasmic proteins in HMVEC-Lung, suggesting that this methodology is compatible with a variety of biochemical and screening assays 20. In summary, this assay provides an easy to use, reproducible, more functional way to access the extent of EC activation by inflammatory mediators in vitro.

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>HMVEC-Lung</td>
 <td>Lonza</td>
 <td>CC-2527</td>
 <td></td>
 </tr>
 <tr>
 <td>EGM-2 MV</td>
 <td>Lonza</td>
 <td>CC-3202</td>
 <td></td>
 </tr>
 <tr>
 <td>HBSS</td>
 <td>Life Technologies</td>
 <td>14175-095</td>
 <td>Can be substituted with any vendor</td>
 </tr>
 <tr>
 <td>48-well Tissue Culture Plates</td>
 <td>BD Falcon</td>
 <td>353078</td>
 <td></td>
 </tr>
 <tr>
 <td>PBS (without phenol red)</td>
 <td>UCSF Cell Culture Facility</td>
 <td>CCFAL001 </td>
 <td>Can be substituted with any vendor</td>
 </tr>
 <tr>
 <td>D-PBS (without Ca2+ and Mg2+ and phenol red)</td>
 <td>UCSF Cell Culture Facility</td>
 <td>CCFAL003</td>
 <td>Can be substituted with any vendor</td>
 </tr>
 <tr>
 <td>RPMI-1640 (without phenol red)</td>
 <td>Life Technologies</td>
 <td>11835-030</td>
 <td></td>
 </tr>
 <tr>
 <td>Polymorphprep</td>
 <td>Axis-Shield</td>
 <td>1114683</td>
 <td></td>
 </tr>
 <tr>
 <td>calcein AM</td>
 <td>Life Technologies</td>
 <td>C3099</td>
 <td>(0.995 mM) stock soln</td>
 </tr>
 <tr>
 <td>Trypan Blue Solution</td>
 <td>Sigma-Aldrich</td>
 <td>T8154</td>
 <td></td>
 </tr>
 <tr>
 <td>40 &mu;M filters</td>
 <td>VWR</td>
 <td>21008-949</td>
 <td>Can be substituted with any vendor</td>
 </tr>
 <tr>
 <td>FLUOstar OPTIMA Spectrophotometer</td>
 <td>BMG LABTECH</td>
 <td>-</td>
 <td></td>
 </tr>
 </tbody>
</table>

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.

In order to obtain reliable, reproducible results using a neutrophil binding assay, it is essential that the health and the confluency of the microvascular endothelial cells are optimal on the day of the assay, as illustrated with HMVEC-Lung in Figure 1. In addition, it is imperative that low passage number microvascular ECs are used (i.e. less than 9 passages), and accordingly, we recommend performing all experiments within two weeks of thawing. The health of the neutrophils is also of utmost i...

Watch this Scientific Journal Video about Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions at JoVE.com

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.

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 ...

quantitative-vitro-assay-to-measure-neutrophil-adhesion-to-activated

Research

JoVE Journal

Immunology and Infection

17.1K Views.  University of California, San Francisco. 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.

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

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

To cause infections, bacteria must colonize their host. Bacterial pathogens express various molecules or structures able to promote attachment to host cells1. These adhesins rely on interactions with host cell surface receptors or soluble proteins acting as a bridge between bacteria and host. Adhesion is a critical first step prior to invasion and/or secretion of toxins, thus it is a key event to be studied in bacterial pathogenesis. Furthermore, adhered bacteria often induce exquisitely fine-tuned cellular responses, the studies of which have given birth to the field of 'cellular microbiology'2. Robust assays for bacterial adhesion on host cells and their invasion therefore play key roles in bacterial pathogenesis studies and have long been used in many pioneer laboratories3,4. These assays are now practiced by most laboratories working on bacterial pathogenesis.
Here, we describe a standard adherence assay illustrating the contribution of a specific adhesin. We use the Escherichia coli strain 27875, a human pathogenic strain expressing the autotransporter Adhesin Involved in Diffuse Adherence (AIDA). As a control, we use a mutant strain lacking the aidA gene, 2787&Delta;aidA (F. Berthiaume and M. Mourez, unpublished), and a commercial laboratory strain of E. coli, C600 (New England Biolabs). The bacteria are left to adhere to the cells from the commonly used HEp-2 human epithelial cell line. This assay has been less extensively described before6.

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

This protocol is a simple bacterial adhesion assay consisting in counting the numbers of bacterial colony forming units that are adhered onto cultured cells. The assay is robust, independent of the adhesin studied, and numerous variations are used in most laboratories working on bacterial pathogenesis.

To cause infections, bacteria must colonize their host. Bacterial pathogens express various molecules or structures able to promote attachment to host ...

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

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

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

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

An In vitro Co-infection Model to Study Plasmodium falciparum-HIV-1 Interactions in Human Primary Monocyte-derived Immune Cells

Plasmodium falciparum, the causative agent of the deadliest form of malaria, and human immunodeficiency virus type-1 (HIV-1) are among the most important health problems worldwide, being responsible for a total of 4 million deaths annually1. Due to their extensive overlap in developing regions, especially Sub-Saharan Africa, co-infections with malaria and HIV-1 are common, but the interplay between the two diseases is poorly understood. Epidemiological reports have suggested that malarial infection transiently enhances HIV-1 replication and increases HIV-1 viral load in co-infected individuals2,3. Because this viremia stays high for several weeks after treatment with antimalarials, this phenomenon could have an impact on disease progression and transmission.
The cellular immunological mechanisms behind these observations have been studied only scarcely. The few in vitro studies investigating the impact of malaria on HIV-1 have demonstrated that exposure to soluble malarial antigens can increase HIV-1 infection and reactivation in immune cells. However, these studies used whole cell extracts of P. falciparum schizont stage parasites and peripheral blood mononuclear cells (PBMC), making it hard to decipher which malarial component(s) was responsible for the observed effects and what the target host cells were4,5. Recent work has demonstrated that exposure of immature monocyte-derived dendritic cells to the malarial pigment hemozoin increased their ability to transfer HIV-1 to CD4+ T cells6,7, but that it decreased HIV-1 infection of macrophages8. To shed light on this complex process, a systematic analysis of the interactions between the malaria parasite and HIV-1 in different relevant human primary cell populations is critically needed.
Several techniques for investigating the impact of HIV-1 on the phagocytosis of micro-organisms and the effect of such pathogens on HIV-1 replication have been described. We here present a method to investigate the effects of P. falciparum-infected erythrocytes on the replication of HIV-1 in human primary monocyte-derived macrophages. The impact of parasite exposure on HIV-1 transcriptional/translational events is monitored by using single cycle pseudotyped viruses in which a luciferase reporter gene has replaced the Env gene while the effect on the quantity of virus released by the infected macrophages is determined by measuring the HIV-1 capsid protein p24 by ELISA in cell supernatants.

An In vitro Co-infection Model to Study Plasmodium falciparum-HIV-1 Interactions in Human Primary Monocyte-derived Immune Cells

We have developed an in vitro malaria-HIV-1 co-infection model to study the impact of Plasmodium falciparum on the HIV-1 replicative cycle in human primary monocyte-derived macrophages. This versatile system can easily be adapted to other primary cell types susceptible to HIV-1 infection.

Plasmodium falciparum, the causative agent  of the deadliest form of malaria, and human immunodeficiency virus type-1 (HIV-1) are among the most important ...

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

Mucosal surfaces serve as protective barriers against pathogenic organisms.&#160;Innate immune responses are activated upon sensing pathogen leading to the infiltration of tissues with migrating inflammatory cells, primarily neutrophils.&#160;This process has the potential to be destructive to tissues if excessive or held in an unresolved state.&#160; Cocultured in vitro models can be utilized to study the unique molecular mechanisms involved in pathogen induced neutrophil trans-epithelial migration.&#160;This type of model provides versatility in experimental design with opportunity for controlled manipulation of the pathogen, epithelial barrier, or neutrophil.&#160;Pathogenic infection of the apical surface of polarized epithelial monolayers grown on permeable transwell filters instigates physiologically relevant basolateral to apical trans-epithelial migration of neutrophils applied to the basolateral surface.&#160;The in vitro model described herein demonstrates the multiple steps necessary for demonstrating neutrophil migration across a polarized lung epithelial monolayer that has been infected with pathogenic P. aeruginosa (PAO1).&#160;Seeding and culturing of permeable transwells with human derived lung epithelial cells is described, along with isolation of neutrophils from whole human blood and culturing of PAO1 and nonpathogenic K12 E. coli (MC1000).&#160; The emigrational process and quantitative analysis of successfully migrated neutrophils that have been mobilized in response to pathogenic infection is shown with representative data, including positive and negative controls.&#160;This in vitro model system can be manipulated and applied to other mucosal surfaces.&#160;Inflammatory responses that involve excessive neutrophil infiltration can be destructive to host tissues and can occur in the absence of pathogenic infections.&#160;A better understanding of the molecular mechanisms that promote neutrophil trans-epithelial migration through experimental manipulation of the in vitro coculture assay system described herein has significant potential to identify novel therapeutic targets for a range of mucosal infectious as well as inflammatory diseases.

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

Neutrophil trans-epithelial migration in response to mucosal bacterial infection contributes to epithelial injury and clinical disease.&#160;An in vitro model has been developed that combines pathogen, human neutrophils, and polarized human epithelial cell layers grown on transwell filters to facilitate investigations towards unraveling the molecular mechanisms orchestrating this phenomenon.

Mucosal surfaces serve as protective barriers against pathogenic organisms.&#160;Innate immune responses are activated upon sensing pathogen leading to ...

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 ...

Static Adhesion Assay for the Study of Integrin Activation in T Lymphocytes

T lymphocyte adhesion is required for multiple T cell functions, including migration to sites of inflammation and formation of immunological synapses with antigen presenting cells. T cells accomplish regulated adhesion by controlling the adhesive properties of integrins, a class of cell adhesion molecules consisting of heterodimeric pairs of transmembrane proteins that interact with target molecules on partner cells or extracellular matrix. The most prominent T cell integrin is lymphocyte function associated antigen (LFA)-1, composed of subunits &#945;L and &#946;2, whose target is the intracellular adhesion molecule (ICAM)-1. The ability of a T cell to control adhesion derives from the ability to regulate the affinity states of individual integrins. Inside-out signaling describes the process whereby signals inside a cell cause the external domains of integrins to assume an activated state. Much of our knowledge of these complex phenomena is based on mechanistic studies performed in simplified in&#160;vitro model systems. The T lymphocyte adhesion assay described here is an excellent tool that allows T cells to adhere to target molecules, under static conditions, and then utilizes a fluorescent plate reader to quantify adhesiveness. This assay has been useful in defining adhesion-stimulatory or inhibitory substances that act on lymphocytes, as well as characterizing the signaling events involved. Although described here for LFA-1 - ICAM-1 mediated adhesion; this assay can be readily adapted to allow for the study of other adhesive interactions (e.g.&#160;VLA-4 - fibronectin).

Static adhesion assay is a powerful tool that can be used to model the interactions between T lymphocytes and other cell types. Interactions are generated by injecting labeled T cells into wells coated with adhesion molecules, while a plate reader is used to quantify the number of adherent cells following serial washes.

T lymphocyte adhesion is required for multiple T cell functions, including migration to sites of inflammation and formation of immunological synapses with ...

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 ...

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.