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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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. 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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 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 ...

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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 ...

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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

Use of an Optical Trap for Study of Host-Pathogen Interactions for Dynamic Live Cell Imaging

Dynamic live cell imaging allows direct visualization of real-time interactions between cells of the immune system1, 2; however, the lack of spatial and temporal control between the phagocytic cell and microbe has rendered focused observations into the initial interactions of host response to pathogens difficult. Historically, intercellular contact events such as phagocytosis3 have been imaged by mixing two cell types, and then continuously scanning the field-of-view to find serendipitous intercellular contacts at the appropriate stage of interaction. The stochastic nature of these events renders this process tedious, and it is difficult to observe early or fleeting events in cell-cell contact by this approach. This method requires finding cell pairs that are on the verge of contact, and observing them until they consummate their contact, or do not. To address these limitations, we use optical trapping as a non-invasive, non-destructive, but fast and effective method to position cells in culture.
Optical traps, or optical tweezers, are increasingly utilized in biological research to capture and physically manipulate cells and other micron-sized particles in three dimensions4. Radiation pressure was first observed and applied to optical tweezer systems in 19705, 6, and was first used to control biological specimens in 19877. Since then, optical tweezers have matured into a technology to probe a variety of biological phenomena8-13.
We describe a method14 that advances live cell imaging by integrating an optical trap with spinning disk confocal microscopy with temperature and humidity control to provide exquisite spatial and temporal control of pathogenic organisms in a physiological environment to facilitate interactions with host cells, as determined by the operator. Live, pathogenic organisms like Candida albicans and Aspergillus fumigatus, which can cause potentially lethal, invasive infections in immunocompromised individuals15, 16 (e.g. AIDS, chemotherapy, and organ transplantation patients), were optically trapped using non-destructive laser intensities and moved adjacent to macrophages, which can phagocytose the pathogen. High resolution, transmitted light and fluorescence-based movies established the ability to observe early events of phagocytosis in living cells. To demonstrate the broad applicability in immunology, primary T-cells were also trapped and manipulated to form synapses with anti-CD3 coated microspheres in vivo, and time-lapse imaging of synapse formation was also obtained. By providing a method to exert fine spatial control of live pathogens with respect to immune cells, cellular interactions can be captured by fluorescence microscopy with minimal perturbation to cells and can yield powerful insight into early responses of innate and adaptive immunity.

A method is described to individually select, manipulate, and image live pathogens using an optical trap coupled to a spinning disk microscope. The optical trap provides spatial and temporal control of organisms and places them adjacent to host cells. Fluorescence microscopy captures dynamic intercellular interactions with minimal perturbation to cells.

Dynamic live cell imaging allows direct visualization of real-time interactions between cells of the immune system1, 2; however, the lack of spatial and ...

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 ...

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

The vascular endothelium plays an integral part in the inflammatory response. During the acute phase of inflammation, endothelial cells (ECs) are activated by host mediators or directly by conserved microbial components or host-derived danger molecules. Activated ECs express cytokines, chemokines and adhesion molecules that mobilize, activate and retain leukocytes at the site of infection or injury. Neutrophils are the first leukocytes to arrive, and adhere to the endothelium through a variety of adhesion molecules present on the surfaces of both cells. The main functions of neutrophils are to directly eliminate microbial threats, promote the recruitment of other leukocytes through the release of additional factors, and initiate wound repair. Therefore, their recruitment and attachment to the endothelium is a critical step in the initiation of the inflammatory response. In this report, we describe an in vitro neutrophil adhesion assay using calcein AM-labeled primary human neutrophils to quantitate the extent of microvascular endothelial cell activation under static conditions. This method has the additional advantage that the same samples quantitated by fluorescence spectrophotometry can also be visualized directly using fluorescence microscopy for a more qualitative assessment of neutrophil binding.

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

Neutrophil adherence to the activated endothelium at sites of infection is an integral component of the host's inflammatory response. Described in this report is a neutrophil binding assay that allows for the in vitro quantitation of primary human neutrophil binding to endothelial cells activated by inflammatory mediators under static conditions.

The vascular  endothelium plays an integral part in the inflammatory response. During the  acute phase of inflammation, endothelial cells (ECs) are ...

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

Leucocyte infiltration into human liver tissue is a common process in all adult inflammatory liver diseases. Chronic infiltration can drive the development of fibrosis and progression to cirrhosis. Understanding the molecular mechanisms that mediate leucocyte recruitment to the liver could identify important therapeutic targets for liver disease. The key interaction during leucocyte recruitment is that of inflammatory cells with endothelium under conditions of shear stress. Recruitment to the liver occurs within the low shear channels of the hepatic sinusoids which are lined by hepatic sinusoidal endothelial cells (HSEC). The conditions within the hepatic sinusoids can be recapitulated by perfusing leucocytes through channels lined by human HSEC monolayers at specific flow rates. In these conditions leucocytes undergo a brief tethering step followed by activation and firm adhesion, followed by a crawling step and subsequent transmigration across the endothelial layer. Using phase contrast microscopy, each step of this &#39;adhesion cascade&#39; can be visualized and recorded followed by offline analysis. Endothelial cells or leucocytes can be pretreated with inhibitors to determine the role of specific molecules during this process.

Leucocyte recruitment to the liver occurs within the specialized channels of the hepatic sinusoids which are lined by unique hepatic sinusoidal endothelial cells. Phase contrast microscopy of leucocyte recruitment across human hepatic sinusoidal endothelium under conditions of physiological shear stress can facilitate the elucidation of the molecular mechanisms which underlie this process.

Leucocyte infiltration into human liver tissue is a common process in all adult inflammatory liver diseases. Chronic infiltration can drive the ...

Human Neutrophil Flow Chamber Adhesion Assay

Neutrophil firm adhesion to endothelial cells plays a critical role in inflammation in both health and disease. The process of neutrophil firm adhesion involves many different adhesion molecules including members of the &beta;2 integrin family and their counter-receptors of the ICAM family. Recently, naturally occurring genetic variants in both &beta;2 integrins and ICAMs are reported to be associated with autoimmune disease. Thus, the quantitative adhesive capacity of neutrophils from individuals with varying allelic forms of these adhesion molecules is important to study in relation to mechanisms underlying development of autoimmunity. Adhesion studies in flow chamber systems can create an environment with fluid shear stress similar to that observed in the blood vessel environment in vivo. Here, we present a method using a flow chamber assay system to study the quantitative adhesive properties of human peripheral blood neutrophils to human umbilical vein endothelial cell (HUVEC) and to purified ligand substrates. With this method, the neutrophil adhesive capacities from donors with different allelic variants in adhesion receptors can be assessed and compared. This method can also be modified to assess adhesion of other primary cell types or cell lines.

A method of quantitating neutrophil adhesion is reported. This method creates a dynamic flow environment similar to that encountered in a blood vessel. It allows the investigation of neutrophil adhesion to either purified adhesion molecules (ligand) or endothelial cell substrate (HUVEC) in a context similar to the in vivo environment with sheer stress.

Neutrophil firm adhesion to endothelial cells plays a critical role in inflammation in both health and disease. The process of neutrophil firm adhesion ...

Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions

Shigella flexneri is an intracellular pathogen that can escape from phagosomes to reach the cytosol, and polymerize the host actin cytoskeleton to promote its motility and dissemination. New work has shown that proteins involved in actin-based motility are also linked to autophagy, an intracellular degradation process crucial for cell autonomous immunity. Strikingly, host cells may prevent actin-based motility of S. flexneri by compartmentalizing bacteria inside &#8216;septin cages&#8217; and targeting them to autophagy. These observations indicate that a more complete understanding of septins, a family of filamentous GTP-binding proteins, will provide new insights into the process of autophagy. This report describes protocols to monitor autophagy-cytoskeleton interactions caused by S. flexneri in vitro using tissue culture cells and in vivo using zebrafish larvae. These protocols enable investigation of intracellular mechanisms that control bacterial dissemination at the molecular, cellular, and whole organism level.

Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions

To counteract pathogen dissemination, host cells reorganize their cytoskeleton to compartmentalize bacteria and induce autophagy. Using Shigella infection of tissue culture cells, host and pathogen determinants underlying this process are identified and characterized. Using zebrafish models of Shigella infection, the role of discovered molecules and mechanisms are investigated in vivo.

Shigella flexneri is an intracellular pathogen that can escape from phagosomes to reach the cytosol, and polymerize the host actin cytoskeleton to promote ...

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 ...

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 ...

Highly Multiplexed, Super-resolution Imaging of T Cells Using madSTORM

Imaging heterogeneous cellular structures using single molecule localization microscopy has been hindered by inadequate localization precision and multiplexing ability. Using fluorescent nano-diamond fiducial markers, we describe the drift correction and alignment procedures required to obtain high precision in single molecule localization microscopy. In addition, a new multiplexing strategy, madSTORM, is described in which multiple molecules are targeted in the same cell using sequential binding and elution of fluorescent antibodies. madSTORM is demonstrated on an activated T cell to visualize the locations of different components within a membrane-bound, multi-protein structure called the T cell receptor microcluster. In addition, application of madSTORM as a general tool for visualization of multi-protein structures is discussed.

We demonstrate a method to image multiple molecules within heterogeneous nano-structures at single molecule accuracy using sequential binding and elution of fluorescently labeled antibodies.

Imaging heterogeneous cellular structures using single molecule localization microscopy has been hindered by inadequate localization precision and ...

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 ...