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 temperature for 1 min with gentle shaking. Neutralize the trypsin with 4 ml of media.</li>
	<li>Count the CHO-ICAM cells using a hemacytometer and calculate 0.75 x 105 cells per chamber. Note: In this experiment: 2.25 x 105 cells will be needed to seed 3 chambers.</li>
	<li>Centrifuge 0.75 x 105 CHO-ICAM cells per chamber for 5 min at 500 x g, room temperature and resuspend the cell pellet in 30 &#181;l per chamber of CHO-ICAM complete culture media. 
	Note: In this experiment 90 &#181;l will be needed to seed 3 chambers.</li>
	<li>Slowly add the cells into one reservoir of the flow chamber. Allow the cells to settle for 5 min in the 37 &#176;C incubator with 5% CO2.</li>
	<li>Add 200 &#181;l of complete culture media across the two reservoirs of each chamber. Alternate additions between the two to avoid movement of the cells as the system equilibrates.</li>
	<li>Incubate the cells overnight inside a 37 &#176;C incubator with 5% CO2.</li>
</ol>
2. Preparation of the T Cells
Note: This assay can be done with primary human T cells or a T cell line. A protocol on T cell isolation from blood has been described previously.10 If using primary human T cells use freshly isolated cells for best results. If using a T cell line, follow the culture protocol specified by the source of the cells. In order to detect the cells on a fluorescent microscope the T cells are labeled with carboxy fluorescein succinimidyl ester (CFSE).
<ol>
	<li>Count the T cells using a hemacytometer and calculate 2 x 106 cells per chamber. Note: In this experiment, 6 x 106 cells will be needed for 3 chambers.</li>
	<li>Centrifuge 2 x 106 T cells per chamber for 5 min at 500 x g, room temperature and resuspend the cells in 1 ml of phosphate buffered saline (PBS) containing 1% glucose or 2% FBS.</li>
	<li>Add CFSE at 1:1,000 dilution (stock made to 5 mM). Cover from light and incubate for 8 min at RT</li>
	<li>Spin at 500 x g for 5 min, RT.</li>
	<li>Resuspend cell pellet with 240 &#181;l per condition (See Section 4 &#34;Note&#34;) of RPMI media lacking serum. In this experiment, resuspend the cell pellet in 720 &#181;l of plain RPMI. Divide the cells into empty 1.5 ml tubes labeled for each chamber, 240 &#181;l per tube. Keep cells in a 37 &#176;C heat block until use.</li>
</ol>
3. Setting up the Input/Output Hosing and Syringe Pump
<ol>
	<li>Warm the microscope live imaging chamber to 37 &#176;C.</li>
	<li>Prepare the input hosing:
	<ol>
		<li>Fill a 500 ml glass bottle with water and make 2 holes into the lid. Label the holes &#34;in&#34; and &#34;out.&#34; This will act as a heating water bath for the input media.</li>
		<li>Run the input hosing through the holes in the lid and into the water bath. Ensure that the side of the hosing that connects to the syringe comes through the &#34;in&#34; hole and the side that will connect to the input reservoir comes through the &#34;out&#34; hole.</li>
		<li>Flush the input hosing with 60 ml of water to remove any air from the system.</li>
		<li>Prime the input hosing with RPMI media lacking serum warmed to 37 &#176;C. Fill a 60 ml syringe and push through the media until the hosing is completely primed and lacking any air bubbles. 
		Note: Depending on the length of tubing used the volume required to prime will vary.
		<ol>
			<li>Calculate the volume of media needed for the experiment by multiplying the rate of flow (here 0.3 ml/min) by the number of minutes (5 min) by the number of chambers in the experiment (here 3); in this experiment, use 4.5 ml media. Note: We recommend having 5 ml extra volume (here totaling 9.5 ml) remaining in the syringe after priming.</li>
		</ol>
		</li>
		<li>Place the entire input setup into the live imaging enclosure of the microscope to maintain 37 &#176;C. Run the hosing and syringe out of the enclosure and load the syringe into the syringe pump.</li>
	</ol>
	</li>
	<li>Prepare the output hosing:
	<ol>
		<li>Make a hole in the lid of a 250 ml bottle to use as output waste.</li>
		<li>Run the output tubing through the hole and into the bottle. Loosely secure the lid, not closing all the way.</li>
		<li>Place the output setup into the live imaging enclosure of the microscope.</li>
	</ol>
	</li>
	<li>Calculate the flow rate (ml/min) necessary to generate the desired shear stress (dyn/cm2). Shear stress varies in different vascular compartments, therefore take into account the overall model including cell types to be studied and treatments when deciding on a shear stress level. 
	Note: These experiments are conducted at 0.4 dyn/cm2 to closely mimic a small vein setting.
	<ol>
		<li>Take the chamber dimensions into account when calculating shear stress. Use the following formula (provided by the manufacturer11) for the flow chambers used here (See Table of Materials): T =&#160;&#951; * 176.1 * &#248; where T = shear stress, &#951; = dynamical viscosity,&#160;&#248; = flow rate. Note: The dynamical viscosity of RPMI media at 37 &#176;C is 0.007.</li>
	</ol>
	</li>
</ol>
4. Loading of the Flow Chambers and Stimulation of the Cells
Note: In order to initiate adhesion, T cells must be stimulated. In the following experiment the cells will either remain unstimulated or are stimulated with SDF-1&#945;12 or phorbol myristate acetate (PMA; positive control)13. For proper presentation of chemokine to T cell, CHO-ICAM cells are preincubated with SDF-1&#945; (followed by serial washes), allowing for presentation on the cell surface. PMA is a phorbol ester that is structurally similar to the second messenger diacyl glycerol (DAG); here it is used as a positive control. T cells are directly treated with PMA prior to loading into the flow chamber.
<ol>
	<li>Place the flow chamber slide on the microscope and set the focal plane on the CHO-ICAM monolayer using the 20X objective.</li>
	<li>Keep CFSE-stained T cells for all other conditions in the 37 &#176;C heat block. Prepare the CHO-ICAM or T cells as follows immediately prior to the assay for that condition:
	<ol>
		<li>For the unstimulated condition (chamber 1), keep one tube/flow chamber untreated, to serve as negative control for stimulation.</li>
		<li>For PMA stimulation (chamber 2), stimulate one tube of T cells with PMA (10 ng/ml) to serve as positive control; treat immediately before loading into flow chamber.</li>
		<li>For SDF-1&#945; stimulation, (chamber 3), stimulate the third flow chamber with SDF-1&#945; (100 ng/ml) by removing 70 &#181;l at a time 3 times from the upper reservoir (output reservoir) and then adding 70 &#181;l of SDF-1&#945; (100 ng/ml) into the lower reservoir (input reservoir). Incubate for 5 min.</li>
	</ol>
	</li>
	<li>Remove and discard 70 &#181;l at a time 3 times from the output reservoir of one chamber.</li>
	<li>Add 70 &#181;l of pre-treated (as applicable) T cell suspension into the input reservoir 3 times. Wait 5 min to allow the cells to move from the inlet of the chamber (&#34;In&#34;), and to reach the proximal outlet (&#34;Out&#34;) of the chamber.</li>
	<li>During this time, image 5 fields for CFSE-stained T cells. Choose fields that are dispersed throughout the center of the chamber (Figure 1). Use the following excitation/emission conditions: excitation laser wavelength: 488 nm; emission collection filter: 500 - 540 nm. These images will serve as the pre-flow cell counts.</li>
	<li>Attach the input and output tubing simultaneously to the flow chamber reservoirs. 
	Note: Attaching the tubing simultaneously is critical so as to not introduce air bubbles into the chamber and maintaining overall equilibrium within the chamber.</li>
	<li>Begin flow at 0.3 ml/min (0.4 dyn/cm2) while visualizing the CFSE-stained T cells. As flow begins, especially with the first chamber, there is often a lag between initiating flow and observing T cell rolling, therefore begin timing 5 min at the onset of T cell movement.</li>
	<li>Terminate flow after 5 min, and image 5 fields in the CFSE channel. Choose fields randomly dispersed throughout the center of the flow chamber (Figure 1). These images will serve as the post-flow cell counts.</li>
</ol>
5. Determining the Percentage of Adherent Cells
Note: Determination of the number of the cells in the acquired images is done objectively with automated software. For our studies we used the software Volocity (Version 6.2.1), but other software such as ImageJ (Version 1.48V) are also applicable.
<ol>
	<li>Determine the number of cells per field pre-flow for each condition. The average of the 5 fields is the &#34;pre-flow average cell count.&#34; 7</li>
	<li>Determine the number of cells per field post-flow for each condition. The average of the 5 fields is the &#34;post-flow average cell count.&#34;7</li>
	<li>Calculate the percentage of adherent cells using the following formula: Percentage of adherent cells = (post-flow average cell count)/(pre-flow average cell count) x 100%.</li>
</ol>

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	<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. O. <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:+getting+a+grip+on+leukocyte+adhesion.">ICAM-1: getting a grip on leukocyte adhesion.</a> J Immunol. 186 (9), 5021-5023 (2011).</li></ol>

The authors have nothing to disclose.

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

assay-adhesion-under-shear-stress-for-study-t-lymphocyte-adhesion

Research

JoVE Journal

Immunology and Infection

8.4K Views.  New York University School of Medicine. 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.

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

Adhesion Frequency Assay for In Situ Kinetics Analysis of Cross-Junctional Molecular Interactions at the Cell-Cell Interface

The micropipette adhesion assay was developed in 1998 to measure two-dimensional (2D) receptor-ligand binding kinetics1. The assay uses a human red blood cell (RBC) as adhesion sensor and presenting cell for one of the interacting molecules. It employs micromanipulation to bring the RBC into contact with another cell that expresses the other interacting molecule with precisely controlled area and time to enable bond formation. The adhesion event is detected as RBC elongation upon pulling the two cells apart. By controlling the density of the ligands immobilized on the RBC surface, the probability of adhesion is kept in mid-range between 0 and 1. The adhesion probability is estimated from the frequency of adhesion events in a sequence of repeated contact cycles between the two cells for a given contact time. Varying the contact time generates a binding curve. Fitting a probabilistic model for receptor-ligand reaction kinetics1 to the binding curve returns the 2D affinity and off-rate.
The assay has been validated using interactions of Fc&gamma; receptors with IgG Fc1-6, selectins with glycoconjugate ligands6-9, integrins with ligands10-13, homotypical cadherin binding14, T cell receptor and coreceptor with peptide-major histocompatibility complexes15-19.
The method has been used to quantify regulations of 2D kinetics by biophysical factors, such as the membrane microtopology5, membrane anchor2, molecular orientation and length6, carrier stiffness9, curvature20, and impingement force20, as well as biochemical factors, such as modulators of the cytoskeleton and membrane microenvironment where the interacting molecules reside and the surface organization of these molecules15,17,19.
The method has also been used to study the concurrent binding of dual receptor-ligand species3,4, and trimolecular interactions19 using a modified model21.
The major advantage of the method is that it allows study of receptors in their native membrane environment. The results could be very different from those obtained using purified receptors17. It also allows study of the receptor-ligand interactions in a sub-second timescale with temporal resolution well beyond the typical biochemical methods.
To illustrate the micropipette adhesion frequency method, we show kinetics measurement of intercellular adhesion molecule 1 (ICAM-1) functionalized on RBCs binding to integrin &alpha;L&beta;2 on neutrophils with dimeric E-selectin in the solution to activate &alpha;L&beta;2.

Adhesion Frequency Assay for In Situ Kinetics Analysis of Cross-Junctional Molecular Interactions at the Cell-Cell Interface

An adhesion frequency assay for measuring receptor-ligand interaction kinetics when both molecules are anchored on the surfaces of the interacting cells is described. This mechanically-based assay is exemplified using a micropipette-pressurized human red blood cell as adhesion sensor and integrin &alpha;L&beta;2 and intercellular adhesion molecule-1 as interacting receptors and ligands.

The micropipette adhesion assay was developed in 1998 to measure two-dimensional (2D) receptor-ligand binding kinetics1. The assay uses a human red blood ...

Bioengineering

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 ...

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 ...

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 ...

Laminar Flow-based Assays to Investigate Leukocyte Recruitment on Cultured Vascular Cells and Adherent Platelets

The recruitment of leukocytes upon injury or inflammation to sites of injury or tissue damage has been investigated during recent decades and has resulted in the concept of the leukocyte adhesion cascade. However, the exact molecular mechanisms involved in leukocyte recruitment have not yet been fully identified. Since leukocyte recruitment remains an important subject in the field of infection, inflammation, and (auto-) immune research, we present a straightforward laminar flow-based assay to study underlying mechanisms of the adhesion, de-adhesion, and transmigration of leukocytes under venous and arterial flow regimes. The in vitro assay can be used to study the molecular mechanisms that underlie the interactions between leukocytes and their cellular partners in different models of vascular inflammation. This protocol describes a laminar flow-based assay using a parallel-flow chamber and an inverted phase contrast microscope connected to a camera to study the interactions of leukocytes and endothelial cells or platelets, which can be visualized and recorded then analyzed offline. Endothelial cells, platelets, or leukocytes can be pretreated with inhibitors or antibodies to determine the role of specific molecules during this process. Shear conditions, i.e. arterial or venous shear stress, can be easily adapted by the viscosity and flow rate of the perfused fluids and the height of the channel.

Leukocytes avidly interact with vascular cells and platelets after vessel wall injury or during inflammation. Here, we describe a straightforward laminar flow-based assay to characterize the molecular mechanisms that underlie the interactions between leukocytes and their cellular partners.

The recruitment of leukocytes upon injury or inflammation to sites of injury or tissue damage has been investigated during recent decades and has resulted ...

Dynamic Adhesion Assay for the Functional Analysis of Anti-adhesion Therapies in Inflammatory Bowel Disease

Gut homing of immune cells is important for the pathogenesis of inflammatory bowel diseases (IBD). Integrin-dependent cell adhesion to addressins is a crucial step in this process and therapeutic strategies interfering with adhesion have been successfully established. The anti-&#945;4&#946;7 integrin antibody, vedolizumab, is used for the clinical treatment of Crohn&#39;s disease (CD) and ulcerative colitis (UC) and further compounds are likely to follow.
The details of the adhesion procedure and the action mechanisms of anti-integrin antibodies are still unclear in many regards due to the limited available techniques for the functional research in this field.
Here, we present a dynamic adhesion assay for the functional analysis of human cell adhesion under flow conditions and the impact of anti-integrin therapies in the context of IBD. It is based on the perfusion of primary human cells through addressin-coated ultrathin glass capillaries with real-time microscopic analysis. The assay offers a variety of opportunities for refinements and modifications and holds potentials for mechanistic discoveries and translational applications.

Dynamic adhesion of immune cells to the vessel wall is a prerequisite for gut homing. Here, we present a protocol for a functional in vitro assay for the impact analysis of anti-integrin antibodies, chemokines or other factors on the dynamic cell adhesion of human cells using addressin-coated capillaries.

Gut homing of immune cells is important for the pathogenesis of inflammatory bowel diseases (IBD). Integrin-dependent cell adhesion to addressins is a ...

Quantitation of Endothelial Cell Adhesiveness In Vitro

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This occurs when endothelial cells, which line blood vessels, become adhesive to circulating immune cells such as monocytes. In vitro measurement of this adhesiveness has until now been done by quantifying the total number of monocytes that adhere to an endothelial layer either as a direct count or by indirect measurement of the fluorescence of adherent monocytes. While such measurements do indicate the average adhesiveness of the endothelial cell population, they are confounded by a number of factors, such as cell number, and do not reveal the proportion of endothelial cells that are actually adhesive. Here we describe and demonstrate a method which allows the enumeration of adhesive cells within a tested population of endothelial monolayer. Endothelial cells are grown on glass coverslips and following desired treatment are challenged with monocytes (that may be fluorescently labeled). After incubation, a rinsing procedure, involving multiple rounds of immersion and draining, the cells are fixed. Adhesive endothelial cells, which are surrounded by monocytes are readily identified and enumerated, giving an adhesion index that reveals the actual proportion of endothelial cells within the population that are adhesive.

Quantitation of Endothelial Cell Adhesiveness In Vitro

We report an in vitro method that allows the quantitation of the actual number of adhesive cells within an endothelial cell monolayer.

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This ...

Biology

Analyzing Cell Surface Adhesion Remodeling in Response to Mechanical Tension Using Magnetic Beads

Mechanosensitive cell surface adhesion complexes allow cells to sense the mechanical properties of their surroundings. Recent studies have identified both force-sensing molecules at adhesion sites, and force-dependent transcription factors that regulate lineage-specific gene expression and drive phenotypic outputs. However, the signaling networks converting mechanical tension into biochemical pathways have remained elusive. To explore the signaling pathways engaged upon mechanical tension applied to cell surface receptor, superparamagnetic microbeads can be used. Here we present a protocol for using magnetic beads to apply forces to cell surface adhesion proteins. Using this approach, it is possible to investigate not only force-dependent cytoplasmic signaling pathways by various biochemical approaches, but also adhesion remodeling by magnetic isolation of adhesion complexes attached to the ligand-coated beads. This protocol includes the preparation of ligand-coated superparamagnetic beads, and the application of define tensile forces followed by biochemical analyses. Additionally, we provide a representative sample of data demonstrating that tension applied to integrin-based adhesion triggers adhesion remodeling and alters protein tyrosine phosphorylation.

Cell surface adhesions are central in mechanotransduction, as they transmit mechanical tension and initiate the signaling pathways involved in tissue homeostasis and development. Here, we present a protocol for dissecting the biochemical pathways that are activated in response to tension, using ligand-coated magnetic microbeads and force application to adhesion receptors.

Mechanosensitive cell surface adhesion complexes allow cells to sense the mechanical properties of their surroundings. Recent studies have identified both ...

A Uniform Shear Assay for Human Platelet and Cell Surface Receptors via Cone-plate Viscometry

Many biological cells/tissues sense the mechanical properties of their local environments via mechanoreceptors, proteins that can respond to forces like pressure or mechanical perturbations. Mechanoreceptors detect their stimuli and transmit signals via a great diversity of mechanisms. Some of the most common roles for mechanoreceptors are in neuronal responses, like touch and pain, or hair cells which function in balance and hearing. Mechanosensation is also important for cell types which are regularly exposed to shear stress such as endothelial cells, which line blood vessels, or blood cells which experience shear in normal circulation. Viscometers are devices that detect the viscosity of fluids. Rotational viscometers may also be used to apply a known shear force to fluids. The ability of these instruments to introduce uniform shear to fluids has been exploited to study many biological fluids including blood and plasma. Viscometry may also be used to apply shear to the cells in a solution, and to test the effects of shear on specific ligand-receptor pairs. Here, we utilize cone-plate viscometry to test the effects of endogenous levels of shear stress on platelets treated with antibodies against the platelet mechanosensory receptor complex GPIb-IX.

We describe an in-solution method to apply uniform shear to platelet surface receptors using cone-plate viscometry. This method may also be used more broadly to apply shear to other cell types and cell-fragments and need not target a specific ligand-receptor pair.

Many biological cells/tissues sense the mechanical properties of their local environments via mechanoreceptors, proteins that can respond to forces like ...

Imaging Molecular Adhesion in Cell Rolling by Adhesion Footprint Assay

Rolling adhesion, facilitated by selectin-mediated interactions, is a highly dynamic, passive motility in recruiting leukocytes to the site of inflammation. This phenomenon occurs in postcapillary venules, where blood flow pushes leukocytes in a rolling motion on the endothelial cells. Stable rolling requires a delicate balance between adhesion bond formation and their mechanically-driven dissociation, allowing the cell to remain attached to the surface while rolling in the direction of flow. Unlike other adhesion processes occurring in relatively static environments, rolling adhesion is highly dynamic as the rolling cells travel over thousands of microns at tens of microns per second. Consequently, conventional mechanobiology methods such as traction force microscopy are unsuitable for measuring the individual adhesion events and the associated molecular forces due to the short timescale and high sensitivity required. Here, we describe our latest implementation of the adhesion footprint assay to image the P-selectin: PSGL-1 interactions in rolling adhesion at the molecular level. This method utilizes irreversible DNA-based tension gauge tethers to produce a permanent history of molecular adhesion events in the form of fluorescence tracks. These tracks can be imaged in two ways: (1) stitching together thousands of diffraction-limited images to produce a large field of view, enabling the extraction of adhesion footprint of each rolling cell over thousands of microns in length, (2) performing DNA-PAINT to reconstruct super-resolution images of the fluorescence tracks within a small field of view. In this study, the adhesion footprint assay was used to study HL-60 cells rolling at different shear stresses. In doing so, we were able to image the spatial distribution of the P-selectin: PSGL-1 interaction and gain insight into their molecular forces through fluorescence intensity. Thus, this method provides the groundwork for the quantitative investigation of the various cell-surface interactions involved in rolling adhesion at the molecular level.

This protocol presents the experimental procedures to perform the adhesion footprint assay to image the adhesion events during fast cell rolling adhesion.

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

Summary

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Chapters in this video

Adhesion Frequency Assay for In Situ Kinetics Analysis of Cross-Junctional Molecular Interactions at the Cell-Cell Interface

Introducing Shear Stress in the Study of Bacterial Adhesion

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

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

Laminar Flow-based Assays to Investigate Leukocyte Recruitment on Cultured Vascular Cells and Adherent Platelets

Dynamic Adhesion Assay for the Functional Analysis of Anti-adhesion Therapies in Inflammatory Bowel Disease

Quantitation of Endothelial Cell Adhesiveness In Vitro

Analyzing Cell Surface Adhesion Remodeling in Response to Mechanical Tension Using Magnetic Beads

A Uniform Shear Assay for Human Platelet and Cell Surface Receptors via Cone-plate Viscometry

Imaging Molecular Adhesion in Cell Rolling by Adhesion Footprint Assay