Name: a flow adhesion assay to study leucocyte recruitment to human hepatic sinusoidal endothelium under conditions of shear stress
Uploaded: 2014-03-21T15:30:00
Duration: 11 min 38 s
Description: Watch this Scientific Journal Video about A Flow Adhesion Assay to Study Leucocyte Recruitment to Human Hepatic Sinusoidal Endothelium Under Conditions of Shear Stress at JoVE.com

S.S. is funded by a Wellcome Trust Intermediate Clinical Fellowship, C.W. by a Wellcome Trust Programme Grant.

1. Microslide Preparation
<ol>
	<li>Precoat a six channel microslide with rat tail collagen type I (RTC, diluted in PBS 1:100 giving a working dilution of 220 &#181;g/ml). This is performed by injecting 30 &#181;l of diluted RTC solution directly into the channels and incubating for 2 hr at 37&#160;&#176;C, followed by three washes with PBS.</li>
</ol>
2. Seeding Cells in Microslides
<ol>
	<li>Dissociate a confluent T75 flask of cultured human hepatic sinusoidal endothelial cells (HSEC) (isolated from liver tissue as described previously13) in trypsin-EDTA, wash in PBS, and resuspend at 3 x 106 cells/ml in complete media (human endothelial basal media supplemented with 2 mM L-glutamine, 100 U/ml&#160;penicillin and 100&#181;g/ml&#160;streptomycin, 10% heat-inactivated AB human serum, 10 ng/ml of vascular endothelial growth factor and 10 ng/ml of hepatocyte growth factor). Inject 30 &#181;l of cell suspension directly into each channel.</li>
	<li>Leave cells to adhere for 1 hr in a humidified incubator at 37 &#176;C with a 5% CO2 atmosphere on a slide rack. After allowing the cells to adhere fill the ports on either side of each channel with complete medium (Figure 1).</li>
</ol>
3. Cytokine Stimulation of Cells
<ol>
	<li>Leave cells in incubator for 24 hr. Assess cell growth using an inverted phase contrast microscope. 24 hr prior to adhesion assay, stimulate the endothelial monolayers with cytokine by replacing the growth media with complete media supplemented by tumor necrosis factor alpha and interferon gamma, both at 10 ng/ml.</li>
</ol>
4. Isolation of Peripheral Blood Lymphocytes
<ol>
	<li>Isolate peripheral blood lymphocytes from whole blood by initially purifying the mononuclear fraction by density gradient centrifugation over an appropriate cell separation centrifugation media for 25 min at 800 x g. Incubate the cells on plastic for 1 hr to allow adherence of monocytes.</li>
	<li>Following adherence to plastic aspirate the lymphocyte enriched supernatant. Wash the lymphocytes and resuspend them (typically 1 x 106 cells/ml in flow medium (endothelial basal media containing 0.1% (v/v) bovine serum albumin (BSA)).</li>
</ol>
5. Pretreatment of Endothelium or Leucocytes with Inhibitors
<ol>
	<li>Prepare recommended dilutions of function blocking antibodies or small molecule inhibitors in endothelial media/0.1% (v/v) BSA. Replace complete medium with blocking antibody/inhibitor solution in chosen microslide channel 30 min prior to flow assay.</li>
	<li>Where pretreatment of chemokine receptors on lymphocytes are planned, resuspend isolated leucocytes in RPMI solution containing 0.1% (v/v) BSA and incubate with 200 ng/ml of Pertussis Toxin to block G-protein coupled receptor activity of chemokine receptors; alternatively dilute specific function blocking antibodies or small molecule inhibitors of chemokine receptors at recommended dilution. Incubate leucocytes at 37 &#176;C for 30 min, then wash and resuspend in flow medium.</li>
</ol>
6. Setup of Flow Assay System
<ol>
	<li>Prewarm a thermostatically controlled transparent chamber to 37 &#176;C. The chamber should have ports for the insertion of silicone tubing and electronic supply for an electronic solenoid valve that enables switching between cells and media with virtually no dead volume. The chamber is mounted on an inverted microscope to allow phase contrast microscopy. Figure 2 demonstrates the flow assay chamber and microscope and microslide placed on the microscope stage.</li>
	<li>Prefill a 50 ml glass syringe with a luer lock with 10 ml of sterile distilled water and attach a length of 25 cm silicone tubing to the syringe port. Insert in a syringe pump. Alter the withdrawal rate of the syringe pump according to the microslide manufacturer&#39;s instructions to maintain a shear stress of 0.05 Pa (0.5 dyne/cm2, Figure 3).</li>
	<li>Take two 5 ml syringes, discard the plungers and attach the barrels to the two in-flow ports of an electronic solenoid valve using silicone tubing.
	<ol>
		<li>Attach 12 cm of silicone tubing to the out-flow valve. The valve permits alternation between a cell free wash buffer (endothelial media/0.1% (v/v) BSA) and the lymphocyte suspension.</li>
		<li>Flush the electronic solenoid valve by inserting wash buffer in both syringe barrels and ensuring buffer is flowing from one barrel through the valve and into the out-flow silicone tubing.</li>
		<li>Use the valve switch to alternate to the other barrel and ensure the buffer is flowing and that all bubbles are removed from the system. Remove wash buffer from one of the barrels and replace with lymphocyte suspension Figure 4a.</li>
	</ol>
	</li>
	<li>Connect the silicone tubing from the syringe pump to one port of a chosen microslide channel via a microslide adaptor. Then connect the silicone tubing from the outflow valve to the opposite port via a microslide adaptor. Ensure that the silicone tubing and the adaptors are filled with wash buffer prior to connection to prevent air bubbles entering the system (Figure 4b).</li>
	<li>Once attached to the flow system, place the microslide on the microscope stage and attach with clips or tape to prevent movement. Set the microscope to the 10X objective together with the appropriate phase setting. Ensure the endothelial monolayer is visualized and in focus using the ocular lens. Ensure images can be captured via a camera which is attached to the microscope whereby images can be relayed to a monitor and recorded.</li>
</ol>
7. Flow Assay Technique and Recording of Adhesion for Analysis
<ol>
	<li>Perfuse the endothelial layer with wash buffer for 2 min by commencing withdrawal of the syringe pump to remove any debris or unbound blocking antibody and then switch the valve to allow a 5 min bolus of leucocyte solution at a constant wall shear stress of 0.05 Pa.</li>
	<li>During the last 2 min of the leucocyte bolus, record 10 random fields along the length of the microslide. This allows offline analysis of lymphocyte rolling/tethering on the endothelial monolayer. Record each field for approximately 10 sec before moving to the next and ensure that recordings are made against the direction of flow to avoid recording the same rolling cell twice in quick succession.</li>
	<li>Follow the leucocyte bolus with a 5 min bolus of wash buffer by returning the valve to its start position. During the final 2 min of this bolus carry out a second recording phase by choosing at least 10 randomly selected fields along the length of the microslide. Record each field for approximately 5 sec before moving to the next and ensure that the endothelial monolayer and adherent leucocytes are in clear focus. This allows offline analysis of the pattern of adhesion.</li>
</ol>

<ol>
	<li>Ley, K., Laudanna, C., Cybulsky, M. I., Nourshargh, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Getting+to+the+site+of+inflammation:+the+leukocyte+adhesion+cascade+updated.">Getting to the site of inflammation: the leukocyte adhesion cascade updated.</a> Nat. Rev. Immunol. 7, 678-689 (2007).</li><li>Wong, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+minimal+role+for+selectins+in+the+recruitment+of+leukocytes+into+the+inflamed+liver+microvasculature.">A minimal role for selectins in the recruitment of leukocytes into the inflamed liver microvasculature.</a> Clin. Invest. , 2782-2790 (1997).</li><li>Braet, F., Wisse, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+and+functional+aspects+of+liver+sinusoidal+endothelial+cell+fenestrae:+a+review.">Structural and functional aspects of liver sinusoidal endothelial cell fenestrae: a review.</a> Compar. Hepatol. 1, 1 (2002).</li><li>Lawrence, M. B., Springer, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leukocytes+roll+on+a+selectin+at+physiologic+flow+rates:+distinction+from+and+prerequisite+for+adhesion+through+integrins.">Leukocytes roll on a selectin at physiologic flow rates: distinction from and prerequisite for adhesion through integrins.</a> Cell. 65, 859-873 (1991).</li><li>Finger, E. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adhesion+through+L-selectin+requires+a+threshold+hydrodynamic+shear.">Adhesion through L-selectin requires a threshold hydrodynamic shear.</a> Nature. 379, 266-269 (1996).</li><li>Lawrence, M. B., Kansas, G. S., Kunkel, E. J., 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=Threshold+levels+of+fluid+shear+promote+leukocyte+adhesion+through+selectins+(CD62L,P,E).">Threshold levels of fluid shear promote leukocyte adhesion through selectins (CD62L,P,E).</a> J. Cell biol. 136, 717-727 (1997).</li><li>Cinamon, G., Shinder, V., Alon, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shear+forces+promote+lymphocyte+migration+across+vascular+endothelium+bearing+apical+chemokines.">Shear forces promote lymphocyte migration across vascular endothelium bearing apical chemokines.</a> Nat. Immunol. 2, 515-522 (2001).</li><li>Lalor, P. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+adhesion+protein-1+mediates+adhesion+and+transmigration+of+lymphocytes+on+human+hepatic+endothelial+cells.">Vascular adhesion protein-1 mediates adhesion and transmigration of lymphocytes on human hepatic endothelial cells.</a> J. Immunol. 169, 983-992 (2002).</li><li>Aspinall, A. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CX(3)CR1+and+vascular+adhesion+protein-1-dependent+recruitment+of+CD16(+)+monocytes+across+human+liver+sinusoidal+endothelium.">CX(3)CR1 and vascular adhesion protein-1-dependent recruitment of CD16(+) monocytes across human liver sinusoidal endothelium.</a> Hepatology. 51, 2030-2039 (2010).</li><li>Curbishley, S. M., Eksteen, B., Gladue, R. P., Lalor, P., Adams, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CXCR+3+activation+promotes+lymphocyte+transendothelial+migration+across+human+hepatic+endothelium+under+fluid+flow.">CXCR 3 activation promotes lymphocyte transendothelial migration across human hepatic endothelium under fluid flow.</a> Am. J. Pathol. 167, 887-899 (2005).</li><li>Oo, Y. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+roles+for+CCR4+and+CXCR3+in+the+recruitment+and+positioning+of+regulatory+T+cells+in+the+inflamed+human+liver.">Distinct roles for CCR4 and CXCR3 in the recruitment and positioning of regulatory T cells in the inflamed human liver.</a> J. Immunol. 184, 2886-2898 (2010).</li><li>Weston, C. J., Shepherd, E. L., Adams, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+localization+and+trafficking+of+vascular+adhesion+protein-1+as+revealed+by+an+N-terminal+GFP+fusion+protein.">Cellular localization and trafficking of vascular adhesion protein-1 as revealed by an N-terminal GFP fusion protein.</a> J. Neural Transm. , (2013).</li><li>Shetty, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Common+lymphatic+endothelial+and+vascular+endothelial+receptor-1+mediates+the+transmigration+of+regulatory+T+cells+across+human+hepatic+sinusoidal+endothelium.">Common lymphatic endothelial and vascular endothelial receptor-1 mediates the transmigration of regulatory T cells across human hepatic sinusoidal endothelium.</a> J. Immunol. 186, 4147-4155 (2011).</li></ol>

The authors declare&#160;no competing financial interests

The most critical step for successfully performing a flow assay is ensuring that a healthy and confluent monolayer of endothelial cells is ready prior to the flow adhesion assay. Primary endothelial cells can be difficult to culture and sensitive to alterations in culturing methods. It is important that 1) flow chambers are adequately and uniformly coated with endothelial cells in a monolayer; for HSEC we use rat tail collagen type I but this may differ for other endothelial populations, 2) culture medium is appropriate for the cell type, for HSEC we have described our complete medium in the protocol section. Other vital steps include setting the syringe pump at the appropriate rate to reflect physiological levels of shear stress.
During the flow assay it is necessary to prevent air bubbles within the flow circuit which can damage the endothelial monolayer or strip immune cells from the endothelial surface. This can be prevented by ensuring that all silicone tubing and adaptors are perfused with wash buffer prior to connection, that all air bubbles are removed and that the media are prewarmed prior to use. When connecting the adaptors to the ports on the microslide it is very important that there is a liquid/liquid interface during connection, if there is any air then this will form an air gap within the system which will disrupt the endothelial monolayer during the syringe withdrawal step. The leucocyte solution in the syringe barrel needs regular agitation to ensure that the cells do not settle, thus maintaining a constant cell density throughout the experiment.
During the recording steps it is important to ensure the image of the endothelial layer is adequately focused and clear to allow accurate offline analysis, and that during the second step of the flow assay (post leucocyte bolus) that enough time is left during the wash buffer phase before recording is recommenced to ensure that all nonadherent leucocytes are removed. Similarly it is essential to use endothelial cells in a monolayer of appropriate density to prevent loss of cells which can interfere with flow patterns in narrow capillaries and also can be hard to discriminate from larger adherent leucocytes under phase contrast microscopy. We have described optimal seeding density for human hepatic sinusoidal endothelial cells but this may vary between different endothelial populations and species.
Significant progress has been made in studying leucocyte recruitment in animal models with intravital microscopy. The major advantage of the flow adhesion assay method is that leucocyte recruitment can be studied in a binary system with primary human endothelial cells. Furthermore, these interactions can be studied under physiological relevant levels of shear stress. It is important to confirm findings of intravital studies in animals with human cellular systems as there may be differences in endothelial properties between species. One of the limitations of the flow assay is that leucocyte recruitment is being studied in a unicellular environment of the endothelial monolayer. In addition once the leucocytes have adhered and transmigrated across the endothelium they may not be in present in sufficient numbers to be isolated and subjected to downstream processes.
Despite these limitations, once the flow adhesion assay has been mastered it can be developed to perform further analysis of the leucocyte adhesion cascade and adapted to recapitulate a multicellular environment. Prolonged recording of single fields and the use of tracking software can be used to analyze crawling behavior of the leucocytes. Furthermore upon completion of the flow assay the microslides can be interrogated using laser scanning confocal microscopy and immunofluorescent labeling to study adhesion and transmigration in more detail. Additionally, we have previously developed an in vitro model where flowing leucocytes could interact with hepatic endothelium conditioned by the presence of hepatocytes. This assay can also be developed to study subpopulations of leucocytes: our group has performed studies with subsets such as regulatory T cells, B cells, and liver infiltrating leucocytes.
These studies are evidence that the flow adhesion assay is a powerful tool to study general and organ specific leucocyte recruitment in human systems.

It is now well established that leucocyte recruitment in general follows the paradigm of the multistep adhesion cascade1. This involves the capture of leucocytes from flowing blood by endothelial cells lining the vessel wall. Initially, leucocytes undergo a rolling step which is mediated by selectin receptors or members of the immunoglobulin superfamily. This allows G-protein coupled receptors (GPCRs) expressed on the leucocyte surface to be activated by chemokines presented on the endothelial glycocalyx. This leads to the alteration of integrin confirmation to a &#39;high affinity&#39; state on the leucocyte surface and arrest and firm adhesion to the endothelium. Firm adhesion is then followed by shape change and crawling of the leucocyte on the vessel. The final step is transmigration through the endothelial monolayer, which can occur via paracellular or transcellular routes.
While the multistep adhesion cascade describes the general mechanism of leucocyte recruitment within the body there are organ specific differences. In the liver the majority of leucocyte recruitment occurs within the hepatic sinusoids in contrast to other organs where recruitment generally occurs within the post-capillary venules2. The hepatic sinusoids are a low shear environment and leucocytes undergo a brief tethering step prior to firm adhesion which is selectin independent2. These channels are lined by the hepatic sinusoidal endothelium which is discontinuous and contains fenestrae, open pores 100-200 nm in diameter, and lack a basement membrane3. Elucidating the molecular mechanisms that mediate leucocyte recruitment across human hepatic sinusoidal endothelium could identify organ specific therapeutic targets for inflammatory liver diseases.
Flow adhesion assays are essential tools in studying leucocyte recruitment. They allow the reconstruction of leucocyte recruitment in presence of shear stress to analyze adhesion under well-defined forces. The most frequent use for the assay is the study leucocyte adhesion to cultured endothelial monolayers or purified substrates. Commercially available flow chambers are used to perfuse cells under conditions of laminar flow between two flat surfaces and then visualize the dynamic process of adhesion on a microscope4. Previous groups have demonstrated that certain adhesive interactions only take place under flow and cannot be studied in static assays5,6.
We have used this technique to recapitulate the hepatic sinusoids and study leucocyte recruitment under conditions of low shear stress. Primary human HSEC are cultured in microslides and leucocytes can then be perfused over this monolayer at a rate of flow calculated to reproduce the shear stress within hepatic sinusoids. The shear stress is a stress that is applied parallel or tangential to a surface as opposed to normal stress which is perpendicular. Any fluid that is moving along a boundary will exert a shear stress on that boundary. Shear stress has been shown to be an essential component of lymphocyte transmigration7. Under these conditions each step of the adhesion cascade can be visualized by phase contrast microscopy. This method has allowed important insights into the recruitment of leucocytes within the liver including the study of conventional adhesion molecules8, the role of chemokines and chemokine receptors9-11,&#160;and atypical adhesion molecules such as the vascular adhesion protein-1 (VAP-1)8,12 and common lymphatic and vascular endothelial receptor-1 (CLEVER-1)13. Whilst this assay has been mentioned in several of our group&#39;s publications, its description has been brief and we have taken this opportunity to provide a detailed step by step guide to help in troubleshooting and prevent technical errors when attempting the assay. Furthermore, we have recently changed the sourcing of microslide chambers which allows accurate alterations in shear stress. We believe this broadens the applicability of the assay to other endothelial and immune cells. The following method describes the preparation and technique for carrying out a flow based adhesion assay with human hepatic sinusoidal endothelial cells and peripheral blood lymphocytes.

<table border="0" cellpadding="0" cellspacing="0" width="1037">
	<colgroup>
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		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Name of the reagent/ Equipment</td>
			<td style="width:168px;">Company</td>
			<td style="width:195px;">Catalog number</td>
			<td style="width:447px;">Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Six channel &#956;-slide VI 0.4 flow chamber&#160;</td>
			<td>Ibidi</td>
			<td>80601</td>
			<td>Other channel size and pre-coated slidess are available depending on assay requirements.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Flow adaptors&#160; &#956;-slide VI 0.4</td>
			<td>Ibidi</td>
			<td>80646</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Flow assay chamber</td>
			<td>Solent Scientific</td>
			<td>33-3322</td>
			<td>These chambers are custom made by the company dpending on the model of microscope and accessories.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Inverted Microscope IX2</td>
			<td>Olympus, UK</td>
			<td>Model IX50</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Harvard Syringe Pump</td>
			<td>Harvard Apparatus, UK</td>
			<td>702101</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Electronic solenoid valve</td>
			<td>Lee Products Limited,UK</td>
			<td>Part Number LFYA1226032H</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Silicon Tubing large</td>
			<td>Fisher Scientific</td>
			<td>FB50855</td>
			<td>2mm Inner diameter, 4mm Outer diameter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Silicone Tubing-small</td>
			<td>Fisher Scientific</td>
			<td>FB50853</td>
			<td>1mm Inner diameter, 3mm Outer diameter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Harvard Glass Syringe</td>
			<td>Harvard Apparatus, UK</td>
			<td>55-0962</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell separation medium/Lympholyte</td>
			<td>VH Bio</td>
			<td>CL-5020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rat Tail Collagen</td>
			<td>Sigma Aldrich</td>
			<td>C3867-1VL</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

This assay has the ability to visualize the multistep flow adhesion cascade and elucidate the underlying molecular mechanisms by comparing results of control experiments to those with molecular inhibitors. Various vascular beds can be recapitulated by incorporating specific endothelial cells and altering shear stress conditions.
Each step of the adhesion cascade can be analyzed offline by following the recording method outlined in the protocol. The first step of the adhesion cascade is the rolling of leucocytes which can be expressed as a percentage of total adherent cells. Offline analysis allows the number of adherent cells to be enumerated in each recorded field during the leucocyte bolus. Playback of the image allows the comparison of cells that are firmly adherent and those that are undergoing a rolling motion across the endothelium. Rolling motion can be visualised using this technique, each field is recorded for at least 10 sec. Rolling cells are identified by their reduced velocity over the endothelial surface compared to flowing cells. This behaviour must be demonstrated for at least 5 sec without detachment. The adhesion cascade within the hepatic sinusoids takes place in a low shear environment and in vivo studies have confirmed minimal rolling with only a brief tethering step. We have confirmed that the flow assay reflects the environment of the hepatic sinusoids by demonstrating that fewer than 10% of adherent leucocytes persistently roll over stimulated HSEC in these assays.
The next step of the adhesion cascade is firm adhesion. Total adherence can be calculated from the second stage of recording during the wash buffer bolus (step 7.3). Offline analysis allows the total number of firmly adherent cells to be counted in each field (Figure 5). Firmly adherent cells are defined as cells that are stationary or shapechanged with slow crawling behavior. The average number of cells per field can then be calculated. This figure can then be used, in conjunction with the total surface area of the field of view (determined using a graticule or equivalent), concentration of lymphocytes (typically 1 x 106 cells/ml) and the flow rate to express the extent of lymphocyte adherence as adherent cells/mm2/106 cells perfused.
Studying the pattern of adhesion involves the analysis of the last two steps of the adhesion cascade including shape-change, crawling and transendothelial migration. Leucocytes adherent to the upper surface of the HSEC monolayer appear phase-bright whilst those that have migrated through the monolayer appear phase dark (Figure 6). The cells can then be classified as exhibiting &#39;static&#39; adhesion (nonmigrated/ round), &#39;shape-changed&#39; morphology or as &#39;migrated&#39; and individual categories are then expressed as a percentage of the total adhesive population.
<img alt="Figure 1" fo:content-width="5in" fo:src="/files/ftp_upload/51330/51330fig1highres.jpg" src="/files/ftp_upload/51330/51330fig1.jpg" /> 
Figure 1. Monolayer of primary human hepatic sinusoidal endothelial cells within flow chamber. A) Microslides filled with media containing monolayer of endothelial cells prior to commencement of flow adhesion assay. B) Phase contrast image of confluent endothelial monolayer, endothelial cells should be seeded in microslide which have been precoated (for human hepatic endothelial cells this should be with rat tail collagen type 1) and it is essential that the endothelial cells are healthy in culture and confluent. <a href="https://www.jove.com/files/ftp_upload/51330/51330fig1highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 2" fo:content-width="5in" fo:src="/files/ftp_upload/51330/51330fig2highres.jpg" src="/files/ftp_upload/51330/51330fig2.jpg" /> 
Figure 2. Flow assay chamber. A flow assay chamber set-up can be seen here, it consists of a transparent chamber which is mounted on an inverted microscope. A heater is placed in the chamber and should be thermostatically controlled to maintain a temperature of 37 &#176;C. There should be ports available to connect silicone tubing from a microslide within the chamber to a syringe pump which is located outside. The microslide is placed directly on the microscope stage. <a href="https://www.jove.com/files/ftp_upload/51330/51330fig1highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 3" fo:content-width="5in" fo:src="/files/ftp_upload/51330/51330fig3highres.jpg" src="/files/ftp_upload/51330/51330fig3.jpg" /> 
Figure 3. Syringe Pump. A syringe pump is connected via silicone tubing to the flow chamber. The pump is set to a specific withdrawal rate depending on the desired shear stress required for the assay. <a href="https://www.jove.com/files/ftp_upload/51330/51330fig1highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 4" fo:content-width="5in" fo:src="/files/ftp_upload/51330/51330fig4highres.jpg" src="/files/ftp_upload/51330/51330fig4.jpg" /> 
Figure 4. Connecting valve to flow chamber. A) An electronic solenoid valve allows switching between two syringe barrels containing either cells or media with virtually no dead space. B) Once the valve is flushed and the two barrels are set up, the silicone tubing from the valve is connected to the flow chamber. It is critical that when connecting the adaptor on the silicone tubing to the port on the flow chamber there is a liquid/liquid interface. <a href="https://www.jove.com/files/ftp_upload/51330/51330fig1highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 5" fo:content-width="5in" fo:src="/files/ftp_upload/51330/51330fig5highres.jpg" src="/files/ftp_upload/51330/51330fig5.jpg" /> 
Figure 5. Measurement of total leucocyte adherence. During the last two minutes of the wash buffer bolus step (as outlined in the protocol), a minimum of ten random fields should be recorded. These can be analyzed off-line and the total number of firmly adherent cells can be counted in each field. Total adhesion of leucocytes can be compared between control chambers and those pretreated with blocking antibodies, here we show a representative field from a control slide and a slide pretreated with intracellular adhesion molecule-1 (ICAM-1) blocking antibody. Arrows have been added to highlight the adherent leukocytes, in the representative field from the control slide there are a total of 25 leukocytes identified and in the ICAM-1 block slide there are a total of 13 leukocytes identified. Scale bars = 100 &#181;m. <a href="https://www.jove.com/files/ftp_upload/51330/51330fig1highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 6" fo:content-width="5in" fo:src="/files/ftp_upload/51330/51330fig6highres.jpg" src="/files/ftp_upload/51330/51330fig6.jpg" /> 
Figure 6. Analysis of the pattern of leucocyte adhesion on endothelial monolayers by phase contrast microscopy. Offline analysis of recorded fields can also be used to study the direction and velocity of leucocyte adhesion. Specific steps of the adhesion cascade can be visualized and quantified using phase contrast imaging. Phase bright cells which are firmly adherent but not activated can be termed &#39;round&#39; adhesion, the cells which are activated and phase bright can be termed &#39;shape changed&#39; and the cells which are phase dark are the cells which have undergone transendothelial migration and can be termed &#39;migrated&#39;. The image shows examples of each pattern of adhesion. <a href="https://www.jove.com/files/ftp_upload/51330/51330fig1highres.jpg" target="_blank">Click here to view larger image</a>.

Watch this Scientific Journal Video about A Flow Adhesion Assay to Study Leucocyte Recruitment to Human Hepatic Sinusoidal Endothelium Under Conditions of Shear Stress at JoVE.com

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

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 ...

a-flow-adhesion-assay-to-study-leucocyte-recruitment-to-human-hepatic

Research

JoVE Journal

Immunology and Infection

Imaging Leukocyte Adhesion to the Vascular Endothelium at High Intraluminal Pressure

Worldwide, hypertension is reported to be in approximately a quarter of the population and is the leading biomedical risk factor for mortality worldwide. In the vasculature hypertension is associated with endothelial dysfunction and increased inflammation leading to atherosclerosis and various disease states such as chronic kidney disease2, stroke3 and heart failure4. An initial step in vascular inflammation leading to atherogenesis is the adhesion cascade which involves the rolling, tethering, adherence and subsequent transmigration of leukocytes through the endothelium. Recruitment and accumulation of leukocytes to the endothelium is mediated by an upregulation of adhesion molecules such as vascular cell adhesion molecule-1 (VCAM-1), intracellular cell adhesion molecule-1 (ICAM-1) and E-selectin as well as increases in cytokine and chemokine release and an upregulation of reactive oxygen species5. In vitro methods such as static adhesion assays help to determine mechanisms involved in cell-to-cell adhesion as well as the analysis of cell adhesion molecules. Methods employed in previous in vitro studies have demonstrated that acute increases in pressure on the endothelium can lead to monocyte adhesion, an upregulation of adhesion molecules and inflammatory markers6 however, similar to many in vitro assays, these findings have not been performed in real time under physiological flow conditions, nor with whole blood. Therefore, in vivo assays are increasingly utilised in animal models to demonstrate vascular inflammation and plaque development. Intravital microscopy is now widely used to assess leukocyte adhesion, rolling, migration and transmigration7-9. When combining the effects of pressure on leukocyte to endothelial adhesion the in vivo studies are less extensive. One such study examines the real time effects of flow and shear on arterial growth and remodelling but inflammatory markers were only assessed via immunohistochemistry10. Here we present a model for recording leukocyte adhesion in real time in intact pressurised blood vessels using whole blood perfusion. The methodology is a modification of an ex vivo vessel chamber perfusion model9 which enables real-time analysis of leukocyte -endothelial adhesive interactions in intact vessels. Our modification enables the manipulation of the intraluminal pressure up to 200 mmHg allowing for study not only under physiological flow conditions but also pressure conditions. While pressure myography systems have been previously demonstrated to observe vessel wall and lumen diameter11 as well as vessel contraction this is the first time demonstrating leukocyte-endothelial interactions in real time. Here we demonstrate the technique using carotid arteries harvested from rats and cannulated to a custom-made flow chamber coupled to a fluorescent microscope. The vessel chamber is equipped with a large bottom coverglass allowing a large diameter objective lens with short working distance to image the vessel. Furthermore, selected agonist and/or antagonists can be utilized to further investigate the mechanisms controlling cell adhesion. Advantages of this method over intravital microscopy include no involvement of invasive surgery and therefore a higher throughput can be obtained. This method also enables the use of localised inhibitor treatment to the desired vessel whereas intravital only enables systemic inhibitor treatment.

This is a method to visualise leukocyte adhesion to the endothelium in harvested pressurised vessels. The technique enables studying vascular adhesion under shear flow with differing intraluminal pressures up to 200 mmHg thus mimic-ing the pathophysiological conditions of high blood pressure.

Worldwide, hypertension is reported to be in approximately a quarter of the population and is the leading biomedical risk factor for mortality worldwide. ...

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

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 Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the phenotypic and functional differences between murine and human monocyte-derived macrophages. Therefore, we here describe an in vitro model to generate and study primary human macrophages. Briefly, after density gradient centrifugation of peripheral blood drawn from a forearm vein, monocytes are isolated from peripheral blood mononuclear cells using negative magnetic bead isolation. These monocytes are then cultured for six days under specific conditions to induce different types of macrophage differentiation or polarization. The model is easy to use and circumvents the problems caused by species-specific differences between mouse and man. Furthermore, it is closer to the in vivo conditions than the use of immortalized cell lines. In conclusion, the model described here is suitable to study macrophage biology, identify disease mechanisms and novel therapeutic targets. Even though not fully replacing experiments with animals or human tissues obtained post mortem, the model described here allows identification and validation of disease mechanisms and therapeutic targets that may be highly relevant to various human diseases.

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Monocyte-derived macrophages are important cells of the innate immune system. Here, we describe an easy to use in vitro model to generate these cells. Using gradient centrifugation, negative bead isolation and specific cell culture conditions, monocyte-derived macrophages can be generated for phenotypic and functional studies.

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the ...

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 ...

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) infections present a broad spectrum of disease severity, ranging from mild infections to life-threatening bronchiolitis. An important part of the pathogenesis of severe disease is an enhanced immune response leading to immunopathology.	Here, we describe a protocol used to investigate the immune response of human immune cells to an HRSV infection. First, we describe methods used for culturing, purification and quantification of HRSV. Subsequently, we describe a human in vitro model in which peripheral blood mononuclear cells (PBMCs) are stimulated with live HRSV. This model system can be used to study multiple parameters that may contribute to disease severity, including the innate and adaptive immune response. These responses can be measured at the transcriptional and translational level. Moreover, viral infection of cells can easily be measured using flow cytometry. Taken together, stimulation of PBMC with live HRSV provides a fast and reproducible model system to examine mechanisms involved in HRSV-induced disease.

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) can cause severe bronchiolitis in young infants. Part of the pathogenesis of severe HRSV disease is caused by the host immune response. Stimulation of primary human immune cells with HRSV provides a fast and reproducible model system to study activation of inflammatory pathways and infection.

Human respiratory syncytial  virus (HRSV) infections present a broad spectrum of disease severity, ranging  from mild infections to life-threatening ...

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 ...

A Simple and Rapid Protocol to Non-enzymatically Dissociate Fresh Human Tissues for the Analysis of Infiltrating Lymphocytes

The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted as an important hallmark of cancer. Our research on breast cancer focuses on the active role that tumor infiltrating lymphocytes play in tumor progression and patient outcome. Toward this goal, we developed a methodology for the rapid isolation of intact lymphoid cells from normal and abnormal tissues in an effort to evaluate them proximate to their native state. Homogenates prepared using a mechanical dissociator show both increased viability and cell recovery while preserving surface receptor expression compared to enzyme-digested tissues. Furthermore, enzymatic digestion of the remaining insoluble material did not recover additional CD45+ cells indicating that quantitative and qualitative measurements in the primary homogenate likely genuinely reflect infiltrating subpopulations in the tissue fragment. The lymphoid cells in these homogenates can be easily characterized using immunological (phenotype, proliferation, etc.) or molecular (DNA, RNA and/or protein) approaches. CD45+ cells can also be used for subpopulation purification, in vitro expansion or cryopreservation. An additional benefit of this approach is that the primary tissue supernatant from the homogenates can be used to characterize and compare cytokines, chemokines, immunoglobulins and antigens present in normal and malignant tissues. This protocol functions extremely well for human breast tissues and should be applicable to a wide variety of normal and abnormal tissues.

This protocol describes the rapid non-enzymatic dissociation of fresh human tissue fragments for qualitative and quantitative assessment of CD45+ cells (lymphocytes/leukocytes) present in various normal and malignant human tissues. Additionally, the supernatant obtained from the primary tissue homogenate can be collected and stored for further analysis or experimentation.

The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted ...

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 ...