The Rheumatology Research Foundation and the Hirschil Trust supported this work.

1. Plating the CHO-ICAM Cells
Note: The goal of this step is to plate the CHO-ICAM cells in the flow chambers for growth overnight with the goal of generating a confluent monolayer.
<ol>
	<li>Maintain CHO-ICAM cells in 10 cm tissue-culture treated culture dishes in 10 ml RPMI media supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (CHO-ICAM complete culture media) at 37 &#176;C with 5% CO2.</li>
	<li>To collect cells, add 1 ml 0.5% trypsin-EDTA and incubate at room temperat...

<ol>
	<li>Alon, R., 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=Cells+on+the+run:+shear-regulated+integrin+activation+in+leukocyte+rolling+and+arrest+on+endothelial+cells.">Cells on the run: shear-regulated integrin activation in leukocyte rolling and arrest on endothelial cells.</a> Curr Opin Cell Biol. 20 (5), 525-532 (2008).</li><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 (9), 678-689 (2007).</li><li>Dustin, M. L., Bivona, T. G., Philips, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membranes+as+messengers+in+T+cell+adhesion+signaling.">Membranes as messengers in T cell adhesion signaling.</a> Nat Immunol. 5 (4), 363-372 (2004).</li><li>Mor, A., Dustin, M. L., Philips, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+GTPases+and+LFA-1+reciprocally+modulate+adhesion+and+signaling.">Small GTPases and LFA-1 reciprocally modulate adhesion and signaling.</a> Immunol Rev. 218, 114-125 (2007).</li><li>Rose, D. M., Alon, R., Ginsberg, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrin+modulation+and+signaling+in+leukocyte+adhesion+and+migration.">Integrin modulation and signaling in leukocyte adhesion and migration.</a> Immunol Rev. 218, 126-134 (2007).</li><li>Alon, R., Feigelson, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemokine-triggered+leukocyte+arrest:+force-regulated+bi-directional+integrin+activation+in+quantal+adhesive+contacts.">Chemokine-triggered leukocyte arrest: force-regulated bi-directional integrin activation in quantal adhesive contacts.</a> Curr Opin Cell Biol. 24 (5), 670-676 (2012).</li><li>Su, W., 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=Rap1+and+its+effector+RIAM+are+required+for+lymphocyte+trafficking.">Rap1 and its effector RIAM are required for lymphocyte trafficking.</a> Blood. 126 (25), 2695-2703 (2015).</li><li>Judokusumo, E., Tabdanov, E., Kumari, S., Dustin, M. L., Kam, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanosensing+in+T+lymphocyte+activation.">Mechanosensing in T lymphocyte activation.</a> Biophys J. 102 (2), L5-L7 (2012).</li><li>Zeltzer, E., 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=Diminished+adhesion+of+CD4++T+cells+from+dialysis+patients+to+extracellular+matrix+and+its+components+fibronectin+and+laminin.">Diminished adhesion of CD4+ T cells from dialysis patients to extracellular matrix and its components fibronectin and laminin.</a> Nephrol Dial Transplant. 12 (12), 2618-2622 (1997).</li><li>Strazza, M., Azoulay-Alfaguter, I., Pedoeem, A., Mor, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Static+adhesion+assay+for+the+study+of+integrin+activation+in+T+lymphocytes.">Static adhesion assay for the study of integrin activation in T lymphocytes.</a> J Vis Exp. (88), (2014).</li><li>Ibidi GmbH. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shear+stress+and+shear+rates+for+ibidi+&#956;-Slides+-+based+on+numerical+calculations.">Shear stress and shear rates for ibidi &#956;-Slides - based on numerical calculations.</a> Application Note 11. , (2014).</li><li>van Gijsel-Bonnello, M., 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=Pantethine+Alters+Lipid+Composition+and+Cholesterol+Content+of+Membrane+Rafts,+With+Down-Regulation+of+CXCL12-Induced+T+Cell+Migration.">Pantethine Alters Lipid Composition and Cholesterol Content of Membrane Rafts, With Down-Regulation of CXCL12-Induced T Cell Migration.</a> J Cell Physiol. 230 (10), 2415-2425 (2015).</li><li>Tozeren, A., 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=Micromanipulation+of+adhesion+of+phorbol+12-myristate-13-acetate-stimulated+T+lymphocytes+to+planar+membranes+containing+intercellular+adhesion+molecule-1.">Micromanipulation of adhesion of phorbol 12-myristate-13-acetate-stimulated T lymphocytes to planar membranes containing intercellular adhesion molecule-1.</a> Biophys J. 63 (1), 247-258 (1992).</li><li>Mortier, A., Van Damme, J., Proost, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overview+of+the+mechanisms+regulating+chemokine+activity+and+availability.">Overview of the mechanisms regulating chemokine activity and availability.</a> Immunol Lett. 145 (1-2), 2-9 (2012).</li><li>Rot, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+binding+assay+for+studying+chemokine+interactions+with+endothelial+cells.">In situ binding assay for studying chemokine interactions with endothelial cells.</a> J Immunol Methods. 273 (1-2), 63-71 (2003).</li><li>Yang, L., 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=ICAM-1+regulates+neutrophil+adhesion+and+transcellular+migration+of+TNF-alpha-activated+vascular+endothelium+under+flow.">ICAM-1 regulates neutrophil adhesion and transcellular migration of TNF-alpha-activated vascular endothelium under flow.</a> Blood. 106 (2), 584-592 (2005).</li><li>Long, E. 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In order to properly analyze T cell adhesion, the stimulant to be included in the study must be considered when choosing an in vitro method. While there are several assays to study signals leading to LFA-1 activation and ICAM-1 binding all methods are not interchangeable. A static adhesion assay10 is best suited to study T cell-APC interactions; alternatively, the shear stress method detailed here is ideal to model T cell-epithelial cell interactions. In vivo, as chemokines are presented alon...

T lymphocyte adhesion mediates a number of distinct processes in a healthy immune system,3 playing critical roles in T cell trafficking and antigen presentation. Whether during immune surveillance or an active immune response these two broad roles for adhesion are critical.4 The physiological signaling events of T cell-endothelial cell interactions are distinct from T cell-antigen presenting cell (APC) interactions, and therefore require distinct methods of study to best understand the signaling cascades involved. The firm adhesion of a T cell to a blood vessel wall during lymphocyte extravasation requires rapid and dynamic integrin activation. The tight interaction between an active state integrin and adhesion molecules along the endothelium leads to adhesion resistant to the flow of blood, allowing T cells to crawl along the surface in search of an area permissive to cell passage.5 The crawling of a T cell bi-directionally along a blood vessel wall is reliant upon polarized adhesion, with a distinct adhesive front end of the T cell.6 Most importantly, firm adhesion and transmigration require resistance to the shear force generated by circulating blood flow.
When designing experiments to study lymphocyte adhesion, attention should be paid to the specific stimulus of interest. While integrin activation is a common and critical component of all forms of T cell adhesion, the cascades of activation are likely to be unique downstream of individual receptors and co-receptors. Likewise, integrin and adhesion molecule pairs function in specialized microenvironments and on specific subpopulations. In this way, these pairs may be regulated quite differently. The model presented here is ideal for the study of signaling cascades leading to integrin activation taking place under shear stress conditions.7 These interactions cannot be adequately understood in a static adhesion system due to the impact these forces have been shown to have directly on T cell behavior.8 Though presented here with T cells and CHO (Chinese Hamster Ovary) cells engineered to express human ICAM-1 (CHO-ICAM cells) the system can easily be modified to study different leukocyte populations or adhesion molecules.
This assay provides a method to quantify T cell adhesiveness and integrin activation using shear stress, providing a model for the firm adhesion stage of leukocyte extravasation. Through the use of CHO-ICAM cells the affinity of LFA-1 for its ligand in live cells can be examined in real time in response to various stimuli of interest. This technique requires easily obtained, commercially available micro-flow chambers in combination with a syringe pump, greatly simplifying the equipment necessary to model blood flow and shear stress as compared to other models.9 Another major advantage of this assay is that specific signaling cascades and the resulting activation state of individual integrins can be cleanly studied through the use of engineered CHO cells expressing human adhesion molecules of interest. Additionally, the combination of quantitative data with live cell imaging is a significant advantage of this method. Overall, while a number of assays of static T cell adhesion have been described that nicely model T cell-APC interactions, these models are insufficient in capturing the dynamic process of T cell-epithelial adhesion. For this reason, when choosing an adhesion assay the stimulus in question must be considered.

<table border="0" cellpadding="0" cellspacing="0" width="950">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:315px;">T cell samples (cell line or primary)</td>
			<td style="width:269px;">ATCC</td>
			<td style="width:119px;">TIB-152</td>
			<td style="width:248px;">Peripheral human T cells</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CHO-ICAM-1 cells</td>
			<td>ATCC</td>
			<td style="width:119px;">CRL-2093</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">&#181;-Slide VI 0.4 ibiTreat</td>
			<td>ibidi</td>
			<td style="width:119px;">80606</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">500 ml glass bottle</td>
			<td>Fisher</td>
			<td style="width:119px;">FB800500</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">250 ml glass bottle</td>
			<td>Fisher</td>
			<td style="width:119px;">FB800250</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Silicone tubing 0.8 mm</td>
			<td>ibidi</td>
			<td style="width:119px;">10841</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Confocal microscope with incubator chamber</td>
			<td>Ziess</td>
			<td style="width:119px;">700</td>
			<td>Any wide field fluorescent microscope</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Syringe pump</td>
			<td>New Era Pump Systems</td>
			<td style="width:119px;">NE-300</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">60 ml syringe</td>
			<td>BD</td>
			<td style="width:119px;">309653</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CFSE</td>
			<td>eBioscience</td>
			<td style="width:119px;">65-0850</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SDF-1&#945;</td>
			<td>R&#38;D</td>
			<td style="width:119px;">350-NS-010/CF</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RPMI</td>
			<td>Lonza</td>
			<td style="width:119px;">12-702F/12</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PBS&#160;</td>
			<td>Lonza</td>
			<td style="width:119px;">17-516F</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microcentrifuge</td>
			<td>Eppendorf&#160;</td>
			<td style="width:119px;">5424</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">D-Glucose</td>
			<td>Sigma Aldrich</td>
			<td style="width:119px;">G8270&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PMA</td>
			<td>Sigma Aldrich</td>
			<td style="width:119px;">16561-29-8</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Volocity software</td>
			<td style="width:269px;">Perkin Elmer</td>
			<td style="width:119px;">Version 6.2.1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ImageJ software</td>
			<td style="width:269px;">NIH</td>
			<td style="width:119px;">Version 1.48V</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tissue-culture treated culture dishes</td>
			<td>Falcon</td>
			<td>353003</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trypsin-EDTA (0.25%) Phenol Red</td>
			<td>Gibco</td>
			<td>25200114</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Heat Inactivated FBS</td>
			<td>Denville</td>
			<td>FB5001-H</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Penicillin/Streptomycin</td>
			<td>Fisher</td>
			<td>BP295950</td>
			<td></td>
		</tr>
	</tbody>
</table>

assay of adhesion under shear stress for the study of t lymphocyte-adhesion molecule interactions

Overall, T cell adhesion is a critical component of function, contributing to the distinct processes of cellular recruitment to sites of inflammation and interaction with antigen presenting cells (APC) in the formation of immunological synapses. These two contexts of T cell adhesion differ in that T cell-APC interactions can be considered static, while T cell-blood vessel interactions are challenged by the shear stress generated by circulation itself. T cell-APC interactions are classified as static in that the two cellular partners are static relative to each other. Usually, this interaction occurs within the lymph nodes. As a T cell interacts with the blood vessel wall, the cells arrest and must resist the generated shear stress.1,2 These differences highlight the need to better understand static adhesion and adhesion under flow conditions as two distinct regulatory processes. The regulation of T cell adhesion can be most succinctly described as controlling the affinity state of integrin molecules expressed on the cell surface, and thereby regulating the interaction of integrins with the adhesion molecule ligands expressed on the surface of the interacting cell. Our current understanding of the regulation of integrin affinity states comes from often simplistic in vitro model systems. The assay of adhesion using flow conditions described here allows for the visualization and accurate quantification of T cell-epithelial cell interactions in real time following a stimulus. An adhesion under flow assay can be applied to studies of adhesion signaling within T cells following treatment with inhibitory or stimulatory substances. Additionally, this assay can be expanded beyond T cell signaling to any adhesive leukocyte population and any integrin-adhesion molecule pair.

Representative results are shown from the flow adhesion assay using Jurkat and primary human CD3+ T cells, as indicated, stimulated with SDF-1&#945;. The negative controls in all shown experiments are unstimulated cells. A threshold basal percent adhesion of unstimulated cells is between 5 - 10%; base adhesion notably above this range indicates a problematic experiment and suggests the start population of T cells were nonspecifically pre-activated in preparation. Fold increase ...

Watch this Scientific Journal Video about Assay of Adhesion Under Shear Stress for the Study of T Lymphocyte-Adhesion Molecule Interactions at JoVE.com

Assay of Adhesion Under Shear Stress for the Study of T Lymphocyte-Adhesion Molecule Interactions

This flow adhesion assay provides a simple, high impact model of T cell-epithelial cell interactions. A syringe pump is used to generate shear stress, and confocal microscopy captures images for quantification. The goal of these studies is to effectively quantify T cell adhesion using flow conditions.

Overall, T cell adhesion is a critical component of function, contributing to the distinct processes of cellular recruitment to sites of inflammation and ...

The overall goal of this assay is to determine the effects of treatments of interest on cellular adhesion under shear stress conditions. This method can help answer key questions in the immune cell signaling field, such as, what factors mediate integrin activation. The main advantage of this technique is that single adhesion molecule integrin pairs can be studied in an easily replicated flow system.Though this method can provide insight into LFA1 activation, it can also be applied to the study of other integrins. Generally, individuals new to this method may struggle because the technical setup must be precisely executed. To harvest the CHO-ICAM cells, treat the culture with 0.5%trypsin EDTA for one minute at room temperature with gentle shaking.When the cells begin to detach, neutralize the trypsin with four milliliters of fresh medium, and count the number of dissociated cells. Dilute the cells to a 0.75 times 10 to the fifth cells per chamber concentration and spin them down in a centrifuge. Next, resuspend the pellet in 30 microliters per flow chamber, in complete CHO-ICAM culture medium, and slowly seed 30 microliter aliquots of cells per reservoir of the flow chamber.Settle the cells in a 37 degrees Celsius incubator with 5%carbon dioxide for five minutes. Then add 200 microliters of complete culture medium into both reservoirs of each chamber, and return the plate to the incubator for overnight culture. After harvesting the T cells, count and dilute the cell suspension to a two times 10 to the sixth cells per chamber concentration.Then centrifuge the cells and resuspend the pellet in one milliliter of PBS, supplemented with 1%glucose per two times ten to the sixth T cells. Next, label the T cells in a 1 to 1000 dilution of CFSE protected from light for eight minutes at room temperature. At the end of the incubation, centrifuge the cells and resuspend the pellet in 240 microliters of serum-free RPMI medium per chamber.Then split the cells into one 1.5 milliliter tube per each chamber, and store the tubes in a 37 degrees Celsius heat block until loading. Before imaging, warm the microscope live imaging chamber to 37 degrees Celsius, and fill a 500 milliliter glass bottle with water. Make two holes in the lid labelled in and out, and run the syringe connecting input hose through the in hole, and the input reservoir connecting hose through the out hole.Use a 60 milliliter syringe to flush the input hosing with 60 milliliters of water, followed by 60 milliliters of serum-free 37 degrees Celsius medium to remove any air from the system. When the hosing is primed, transfer the entire input setup into the live imaging chamber of the microscope to maintain the system at 37 degrees Celsius. Run the hosing and syringe out of the chamber, and load the syringe into the syringe pump.Next, make a hole in the lid of a 250 milliliter bottle to serve as the output waste container and run the output tubing through the hole into the bottle. Then loosely secure the output bottle lid and place the entire output setup into the live imaging chamber. Now place the flow chamber slide onto the microscope stage, and use the 20X objective to set the focal plane on the CHO ICAM mono layer.Then, beginning with the unstimulated control, discard three 70 microliter volumes from the output reservoir of the first chamber, and add three 70 microliter volumes of CFCS labeled T cells to the input reservoir. While the T cells are moving from the inlet to the outlet, image five random fields of CFSE positive T cells in each chamber to obtain the pre flow cell counts. Next, simultaneously attach the input and output hoses to the flow chamber reservoirs, taking care not to introduce air bubbles, and begin the shear stress flow at 0.3 milliliters per minute, while continuing to visuals the CFSC stained T cells.As soon as movement of the T cells is observed, start a timer for five minutes, terminating the flow at the end of the five minute period. Then, image five fields of CFSE positive cells in each chamber, choosing the fields randomly to obtain the post flow cell counts. For PMA stimulation, stimulate one tube of T cells with PMA to serve as the positive control immediately before loading the T cells into the second flow chamber for imaging, as just demonstrated.For SDF-1 alpha stimulation, first remove three 70 microliter aliquots of medium from the output reservoir of the third chamber, and then add 70 microliters of SDF-1 alpha into the input reservoir. After five minutes, discard three 70 microliter aliquots from the output reservoir, and add the remaining tube of T cells for imaging, as just demonstrated. In these representative images, a similar number of T cells are observed pre flow under the different stimulation conditions.Following five minutes of continuous shear stress, the number of adherent T cells increases under the PMA and SDF-1 alpha stimulation conditions, while a few unstimulated T cells remain adhered. Indeed, SDF-1 alpha T cell activation, for example, induces a near two-fold increase in T cell adhesion, compared to the unstimulated control cells. Further, when the percent adhesion of primary human CD3 positive T cells in the presence or absence of SDF-1 alpha is calculated, 15%of the unstimulated T cells adhere under shear stress, compared to 50%of the T cells under SDF-1 alpha activation conditions.Once mastered, this technique can be reasonably expanded to six experimental chambers in one assay. When attempting this procedure, it's important to remember to work only with the confluential ICAM mono layer. Following this procedure, other methods, such as cell shape analysis can be performed on the captured images to answer additional questions.After watching this video, you should have a good understanding of how to quantify cell adhesion under shear stress condition. Thank you for watching this video, and good luck with your experiments.

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8.3K visualizações.  New York University School of Medicine. The overall goal of this assay is to determine the effects of treatments of interest on cellular adhesion under shear stress conditions. This method can help answer key questions in the immune cell signaling field, such as, what factors mediate integrin activation. The main advantage of this technique is that single adhesion molecule integrin pairs can be studied in an easily replicated flow system.Though this method can provide insight into LFA1 activation, it can also be applied to t...