This work was supported by the Canada Foundation of Innovation (CFI 35492), Natural Sciences and Engineering Research Council of Canada Discovery Grant (RGPIN-2017-04407), New Frontiers in Research Fund (NFRFE-2018-00969), Michael Smith Foundation for Health Research (SCH-2020-0559), and the University of British Columbia Eminence Fund.

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The authors declare no conflict of interest.

The adhesion footprint assay enables visualization of the molecular adhesion events between PSGL-1 and P-selectin during cell rolling adhesion. This process is initiated by P-selectin-mediated capturing followed by rolling under fluidic shear stress. Potential issues during the experiment usually involve poor cell rolling or missing fluorescent tracks even when cells roll well. These problems are often resulting from quality controls at the critical steps in the protocol, as listed in the troubleshooting table (T...

The rolling adhesion cascade describes how circulating cells tether to and roll along the blood vessel wall1. Passive rolling is primarily mediated by selectins, a major class of cellular adhesion molecules (CAMs)1. Under the shear flow of blood, leukocytes expressing P-selectin glycoprotein ligand-1 (PSGL-1) form highly transient bonds with P-selectin, which may be expressed on the surface of inflamed endothelial cells. This process is critical for leukocytes to migrate to a site of inflammation2. In addition, PSGL-1 is also a mechanosensitive receptor capable of triggering the subsequent firm adhesion stage of the rolling adhesion cascade upon its engagement with P-selectin3.
Genetic mutations affecting CAM function can severely affect the immune system, such as in the rare disease of leukocyte adhesion deficiency (LAD), where malfunction of adhesion molecules mediating rolling leads to severely immunocompromised individuals4,5,6. In addition, circulating tumor cells have been shown to migrate following a similar rolling process, leading to metastasis7,8. However, because cell rolling is fast and dynamic, conventional experimental mechanobiology methods are unsuitable for studying molecular interactions during cell rolling. While single-cell and single-molecule manipulation methods like atomic force microscopy and optical tweezer were able to study molecular interactions such as P-selectin&#39;s force-dependent interaction with PSGL-1 at the single-molecule level9, they are unsuitable for investigating live adhesion events during cell rolling. Additionally, the interaction characterized in vitro cannot directly answer the question about molecular adhesion in vivo. For instance, what molecular tension range is biologically relevant when cells are functioning in their native environment? Computational methods such as adhesive dynamics simulation10 or simple steady-state model11 have captured certain molecular details and how they influence the rolling behavior but are highly dependent on the accuracy of the modeling parameters and assumptions. Other techniques such as traction force microscopy can detect forces during cell migration but do not provide sufficient spatial resolution or quantitative information on molecular tension. None of these techniques can provide direct experimental observations of the temporal dynamics, spatial distribution, and magnitude heterogeneity of molecular forces, which directly relate to cell function and behavior in their native environment.
Therefore, implementing a molecular force sensor capable of accurately measuring selectin-mediated interactions is crucial to improving our understanding of rolling adhesion. Here, we describe the protocol for the adhesion footprint assay12 where PSGL-1 coated beads are rolled on a surface presenting p-selectin functionalized tension gauge tethers (TGTs)13. These TGTs are irreversible DNA-based force sensors that result in a permanent history of rupture events in the form of fluorescence readout. This is achieved through the rupturing of the TGT (dsDNA) and then subsequent labeling of the ruptured TGT (ssDNA) with a fluorescently labeled complementary strand. One major advantage of this system is its compatibility with both diffraction-limited and super-resolution imaging. The fluorescently labeled complementary strand can either be permanently bound (&#62;12 bp) for diffraction-limited imaging or transiently bound (7-9 bp) for super-resolution imaging through DNA PAINT. This is an ideal system to study rolling adhesion as the TGTs are ruptured during active rolling, but the fluorescence readout is analyzed post-rolling. The two imaging methods also provide the user with more freedom to investigate rolling adhesion. Typically, diffraction-limited imaging is useful for extracting molecular rupture force through fluorescence intensity13, whereas super-resolution imaging allows for quantitative analysis of receptor density. With the ability to investigate these properties of rolling adhesion, this approach provides a promising platform for understanding the force-regulation mechanism on the molecular adhesion of rolling cells under shear flow.

<table><tbody><tr><td>4-channel drill guide</td><td>Custom made</td><td></td><td>3D printed with ABS filament</td></tr><tr><td>4-holes slide</td><td>Custom made</td><td></td><td>Drill clean microscope slide using a Dremel with diamond coated drill bits on a 4-channels drill guide which has a layout that matches with the centers of the 8-32 threaded holes on the aluminum clamp.</td></tr><tr><td>Acetone</td><td>VWR</td><td>BDH1101-4LP</td><td></td></tr><tr><td>Acrylic spacer</td><td>Custom made</td><td></td><td>Cut two blocks of acrylic sheets with the dimension of 40 mm x 30 mm x 2.5 mm. On each block, drill two 3 mm holes that are precisely aligned with the 4-40 holes on the aluminium holder.</td></tr><tr><td>Aluminium chip holder</td><td>Custom made</td><td></td><td>Machine anodized aluminium block into a C-shaped holder with the outer dimension of 640 mm x 500 mm x 65 mm and the opening dimension of 400 mm x 380 mm x 65 mm. Inlets and outlets are tapped with 8-32 thread.</td></tr><tr><td>Aminosilane</td><td>AlfaAesar</td><td>L14043</td><td>CAS 1760-24-3</td></tr><tr><td>Antibiotic/antimycotic solution</td><td>Cytiva HyClone</td><td>SV3007901</td><td>Pen/Strep/Fungiezone</td></tr><tr><td>Beads, ProtG coated polystyrene</td><td>Spherotech</td><td>PGP-60-5</td><td></td></tr><tr><td>Bovine serum albumin</td><td>VWR</td><td>332</td><td></td></tr><tr><td>Buffer, DNA PAINT</td><td></td><td></td><td>0.05% Tween-20, 5 mM Tris, 75 mM MgCl&lt;sub&gt;2&lt;/sub&gt;, 1 mM EDTA</td></tr><tr><td>Buffer, T50M5</td><td></td><td></td><td>10mM Tris, 50 mM NaCl, 5 mM MgCl&lt;sub&gt;2&lt;/sub&gt;</td></tr><tr><td>Buffer, Rolling</td><td></td><td></td><td>HBSS with 2mM CaCl&lt;sub&gt;2&lt;/sub&gt;, 2 mM MgCl&lt;sub&gt;2&lt;/sub&gt;, 10 mM Hepes, 0.1% BSA</td></tr><tr><td>Buffer, Wash</td><td></td><td></td><td>10 mM Tris, 50 mM NaCl, 5 mM MgCl&lt;sub&gt;2&lt;/sub&gt; and 2 mM CaCl&lt;sub&gt;2&lt;/sub&gt;, 0.05% Tween 20</td></tr><tr><td>Calcium chloride</td><td>VWR</td><td>BDH9224</td><td></td></tr><tr><td>Cell culture flasks</td><td>VWR</td><td>10062-868</td><td></td></tr><tr><td>Concentrated sulfuric acid</td><td>VWR</td><td>BDH3072-2.5LG</td><td>95-98%</td></tr><tr><td>Coverslip holding tweezers</td><td>Techni-Tool</td><td>758TW150</td><td></td></tr><tr><td>Diamond-coated drill bits</td><td>Abrasive technology</td><td>C5250510</td><td>0.75 mm diamond drill</td></tr><tr><td>DNA, amine-ssDNA (top strand)</td><td>IDT DNA</td><td>Custom oligo</td><td>CCGGGCGACGCAGGAGGG /3AmMO/</td></tr><tr><td>DNA, biotin-ssDNA (bottom strand)</td><td>IDT DNA</td><td>Custom oligo</td><td>/5BiotinTEG/ TTTTT CCCTCCTGCGTCGCCCGG</td></tr><tr><td>DNA, imager strand for DNA-PAINT</td><td>IDT DNA</td><td>Custom oligo</td><td>GAGGGAAA TT/3Cy3Sp/</td></tr><tr><td>DNA, imager strand for permanent labelling</td><td>IDT DNA</td><td>Custom oligo</td><td>CCGGGCGACGCAGG /3Cy3Sp/</td></tr><tr><td>Double-sided tape</td><td>Scotch</td><td>237</td><td>3/4 inch width, permanent double-sided tape</td></tr><tr><td>EDTA</td><td>Thermofisher</td><td>15575020</td><td>0.5 M EDTA, pH 8.0</td></tr><tr><td>Epoxy</td><td>Gorilla</td><td>42001</td><td>5 minute curing time</td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Avantor</td><td>97068-085</td><td></td></tr><tr><td>GelGreen</td><td>Biotium</td><td>41005</td><td></td></tr><tr><td>Glacial acetic acid</td><td>VWR</td><td>BDH3094-2.5LG</td><td></td></tr><tr><td>Glass, Coverslips</td><td>Fisher Scientific</td><td>12-548-5P</td><td></td></tr><tr><td>Glass, Microscope slide</td><td>VWR</td><td>48300-026</td><td>75 mm x 25 mm x 1 mm</td></tr><tr><td>Glass, Staining jar</td><td>VWR</td><td>74830-150</td><td>Wheaton Staining Jar (900620)</td></tr><tr><td>Hanks&#039; Balanced Salt solution (HBSS)</td><td>Lonza</td><td>04-315Q</td><td></td></tr><tr><td>Hemocytometer</td><td>Sigma-Aldrich</td><td>Z359629-1EA</td><td></td></tr><tr><td>HL-60 cells</td><td>ATCC</td><td>CCL-240</td><td></td></tr><tr><td>Humidity chamber slide support</td><td>Custom made</td><td></td><td>3D printed with ABS filament</td></tr><tr><td>Hydrogen peroxide</td><td>VWR</td><td>BDH7690-1</td><td>30%</td></tr><tr><td>Imidazole</td><td>Sigma-Aldrich</td><td>I2399</td><td></td></tr><tr><td>Inlets/outlets</td><td>Custom made</td><td></td><td>Drill through eight 8-32 set screws using cobalt drill bits. Insert 1.5 cm&amp;nbsp; polyethylene tubing (Tygon, I.D. 1/32&amp;rdquo; O.D. 3/32&amp;rdquo;) into each hollow setscrew</td></tr><tr><td>Iscove Modified Dulbecco Media (IMDM)</td><td>Lonza</td><td>12-722F</td><td></td></tr><tr><td>Magnesium chloride</td><td>VWR</td><td>BDH9244</td><td></td></tr><tr><td>Magnetic Ni-NTA beads</td><td>Invitrogen</td><td>10103D</td><td></td></tr><tr><td>Mailer tubes</td><td>EMS</td><td>EMS71406-10</td><td></td></tr><tr><td>Methanol</td><td>VWR</td><td>BDH1135-4LP</td><td></td></tr><tr><td>Micro Bio-Spin P-6 Gel Columns</td><td>Biorad</td><td>7326200</td><td>In SSC Buffer</td></tr><tr><td>PEG</td><td>Laysan Bio</td><td>MPEG-SVA-5000</td><td></td></tr><tr><td>PEG-biotin</td><td>Laysan Bio</td><td>Biotin-PEG-SVA-5000</td><td></td></tr><tr><td>Potassium hydroxide</td><td>VWR</td><td>470302-132</td><td></td></tr><tr><td>Protein, Protein G</td><td>Abcam</td><td>ab155724</td><td>N-terminal His-Tag and C-terminal cysteine</td></tr><tr><td>Protein, P-selectin-Fc</td><td>R&amp;amp;D System</td><td>137-PS</td><td>Recombinant Human P-Selectin/CD62P Fc Chimera Protein, CF</td></tr><tr><td>Protein, Streptavidin</td><td>Cedarlane</td><td>CL1005-01-5MG</td><td></td></tr><tr><td>Pump Syringe</td><td>Harvard Apparatus</td><td>704801</td><td></td></tr><tr><td>Sodium bicarbonate</td><td>Ward&amp;rsquo;s Science</td><td>470302-444</td><td></td></tr><tr><td>Sodium chloride</td><td>VWR</td><td>97061-274</td><td></td></tr><tr><td>Sulfo-SMCC</td><td>Thermofisher</td><td>22322</td><td></td></tr><tr><td>Syringe</td><td>Hamilton</td><td>81520</td><td>Syringes with PTFE luer lock, 5 mL</td></tr><tr><td>Syringe needles</td><td>BD</td><td>305115</td><td>Precision Glide 26 G, 5/8 Inch Length</td></tr><tr><td>TCEP</td><td>Sigma-Aldrich</td><td>C4706-2G</td><td></td></tr><tr><td>Tris</td><td>VWR</td><td>BDH4502-500GP</td><td></td></tr><tr><td>Tubing, Adaptor</td><td>Tygon</td><td>ABW00001</td><td>Formulation 3350, I.D. 1/32&amp;rdquo;; O.D. 3/32&amp;rdquo;</td></tr><tr><td>Tubing, Polyethylene</td><td>BD Intramedic</td><td>427406</td><td>Intramedic (PE20) I.D. 0.38mm; O.D. 1.09mm</td></tr><tr><td>Tween-20</td><td>Sigma-Aldrich</td><td>93773</td><td></td></tr></tbody></table>

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.

The protocol above describes the experimental procedure of the adhesion footprint assay. The general experiment workflow is illustrated in Figure 1, from the flow chamber assembly (Figure 1A) to the surface functionalization (Figure 1B) and experiment and imaging steps (Figure 1C).
Figure 2 is a representative result for the ProtG-ssDNA bioconjug...

Watch this Scientific Journal Video about Imaging Molecular Adhesion in Cell Rolling by Adhesion Footprint Assay at JoVE.com

Imaging Molecular Adhesion in Cell Rolling by Adhesion Footprint Assay

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

Rolling adhesion, facilitated by selectin-mediated interactions, is a highly dynamic, passive motility in recruiting leukocytes to the site of ...

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3.1K Views.  The University of British Columbia. Dit protocol helpt bij het leggen van het verband tussen moleculaire kracht en celrolgedrag, naast het ruimtelijk in kaart brengen en kwantificeren van rollende adhesie op moleculair niveau. Deze techniek maakt het mogelijk om individuele adhesiegebeurtenissen tijdens het eigenlijke celrollen te bestuderen, waardoor we de fysiologisch relevante moleculaire adhesiekracht direct kunnen meten. Oppervlaktevoorbereiding met behulp van de juiste concentratie en incubatietijd in elke stap is cruciaa...

Video: Imaging Molecular Adhesion in Cell Rolling by Adhesion Footprint Assay

Studying Cell Rolling Trajectories on Asymmetric Receptor Patterns

Lateral displacement of cells orthogonal to a flow stream by rolling on asymmetric receptor patterns presents an opportunity for development of new devices for label-free separation and analysis of cells1. Such devices may use lateral displacement for continuous-flow separation, or receptor patterns that modulate adhesion to distinguish between different cell phenotypes or levels of receptor expression. Understanding the nature of cell rolling trajectories on receptor-patterned substrates is necessary for engineering of the substrates and design of such devices.
Here, we demonstrate a protocol for studying cell rolling trajectories on asymmetric receptor patterns that support cell rolling adhesion2. Well-defined, &mu;m-scale patterns of P-selectin receptors were fabricated using microcontact printing on gold-coated slides that were incorporated in a flow chamber. HL60 cells expressing the PSGL-1 ligand 3were flowed across a field of patterned lines and visualized on an inverted bright field microscope. The cells rolled and tracked along the inclined edges of the patterns, resulting in lateral deflection1. Each cell typically rolled for a certain distance along the pattern edges (defined as the edge tracking length), detached from the edge, and reattached to a downstream pattern. Although this detachment makes it difficult to track the entire trajectory of a cell from entrance to exit in the flow chamber, particle-tracking software was used to analyze and yield the rolling trajectories of the cells during the time when they were moving on a single receptor-patterned line. The trajectories were then examined to obtain distributions of cell rolling velocities and the edge tracking lengths for each cell for different patterns.
This protocol is useful for quantifying cell rolling trajectories on receptor patterns and relating these to engineering parameters such as pattern angle and shear stress. Such data will be useful for design of microfluidic devices for label-free cell separation and analysis.

We describe a protocol to observe and analyze cell rolling trajectories on asymmetric receptor-patterned substrates. The resulting data are useful for engineering of receptor-patterned substrates for label-free cell separation and analysis.

Lateral displacement of cells orthogonal to a flow stream by rolling on asymmetric receptor patterns presents an opportunity for development of new ...

Bioengineering

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 ...

Creating Adhesive and Soluble Gradients for Imaging Cell Migration with Fluorescence Microscopy

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as growth factors. Here, we outline a method to create opposing gradients of adhesive and soluble cues in a microfluidic chamber, which is compatible with live cell imaging. A copolymer of poly-L-lysine and polyethylene glycol (PLL-PEG) is employed to passivate glass coverslips and prevent non-specific adsorption of biomolecules and cells. Next, microcontact printing or dip pen lithography are used to create tracks of streptavidin on the passivated surfaces to serve as anchoring points for the biotinylated peptide arginine-glycine-aspartic acid (RGD) as the adhesive cue. A microfluidic device is placed onto the modified surface and used to create the gradient of adhesive cues (100% RGD to 0% RGD) on the streptavidin tracks. Finally, the same microfluidic device is used to create a gradient of a chemoattractant such as fetal bovine serum (FBS), as the soluble cue in the opposite direction of the gradient of adhesive cues.

A method for the assembly of adhesive and soluble gradients in a microscopy chamber for live cell migration studies is described. The engineered environment combines antifouling surfaces and adhesive tracks with solution gradients and therefore allows one to determine the relative importance of guidance cues.

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as ...

Systematic Analysis of In Vitro Cell Rolling Using a Multi-well Plate Microfluidic System

A major challenge for cell-based therapy is the inability to systemically target a large quantity of viable cells with high efficiency to tissues of interest following intravenous or intraarterial infusion. Consequently, increasing cell homing is currently studied as a strategy to improve cell therapy. Cell rolling on the vascular endothelium is an important step in the process of cell homing and can be probed in-vitro using a parallel plate flow chamber (PPFC). However, this is an extremely tedious, low throughput assay, with poorly controlled flow conditions. Instead, we used a multi-well plate microfluidic system that enables study of cellular rolling properties in a higher throughput under precisely controlled, physiologically relevant shear flow1,2. In this paper, we show how the rolling properties of HL-60 (human promyelocytic leukemia) cells on P- and E-selectin-coated surfaces as well as on cell monolayer-coated surfaces can be readily examined. To better simulate inflammatory conditions, the microfluidic channel surface was coated with endothelial cells (ECs), which were then activated with tumor necrosis factor-&#945; (TNF-&#945;), significantly increasing interactions with HL-60 cells under dynamic conditions. The enhanced throughput and integrated multi-parameter software analysis platform, that permits rapid analysis of parameters such as rolling velocities and rolling path, are important advantages for assessing cell rolling properties in-vitro. Allowing rapid and accurate analysis of engineering approaches designed to impact cell rolling and homing, this platform may help advance exogenous cell-based therapy.

Systematic Analysis of In Vitro Cell Rolling Using a Multi-well Plate Microfluidic System

This study used a multi-well plate microfluidic system, significantly increasing throughput of cell rolling studies under physiologically relevant shear flow. Given the importance of cell rolling in the multi-step cell homing cascade and the importance of cell homing following systemic delivery of exogenous populations of cells in patients, this system offers potential as a screening platform to improve cell-based therapy.

A major challenge for cell-based therapy is the inability to systemically target a large quantity of viable cells with high efficiency to tissues of ...

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 ...

Immunology and Infection

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.

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 ...

Visualizing Adhesion Formation in Cells by Means of Advanced Spinning Disk-Total Internal Reflection Fluorescence Microscopy

In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to visualize these highly dynamic events, two complementary light microscopy techniques that allow fast imaging of live samples were combined: spinning disk microscopy (SD) for fast and high-resolution volume recording and total internal reflection fluorescence (TIRF) microscopy for precise localization and visualization of the plasma membrane. A comprehensive and complete imaging protocol will be shown for guiding through sample preparation, microscope calibration, image formation and acquisition, resulting in multi-color SD-TIRF live imaging series with high spatio-temporal resolution. All necessary image post-processing steps to generate multi-dimensional live imaging datasets, i.e. registration and combination of the individual channels, are provided in a self-written macro for the open source software ImageJ. The imaging of fluorescent proteins during initiation and maturation of adhesion complexes, as well as the formation of the actin cytoskeletal network, was used as a proof of principle for this novel approach. The combination of high resolution 3D microscopy and TIRF provided a detailed description of these complex processes within the cellular environment and, at the same time, precise localization of the membrane-associated molecules detected with a high signal-to-background ratio.

An advanced microscope that permit fast and high-resolution imaging of both, the isolated plasma membrane and the surrounding intracellular volume, will be presented. The integration of spinning disk and total internal reflection fluorescence microscopy in one setup allows live imaging experiments at high acquisition rates up to 3.5 s per image stack.

In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to ...

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 ...

Quantification of Cell-Substrate Adhesion Area and Cell Shape Distributions in MCF7 Cell Monolayers

The methods presented here quantify some parameters of confluent adherent cell monolayers from multiple appropriately stained confocal images: adhesion to the substrate as a function of the number and size of focal adhesions, and cell shape, characterized by the cell shape index and other shape descriptors. Focal adhesions were visualized by paxillin staining and cell-cell borders were marked by junction plakoglobin and actin. The methods for cell culture and staining were standard; images represent single focal planes; image analysis was performed using publicly available image processing software. The presented protocols are used to quantify the number and size of focal adhesions and the differences in cell shape distribution in the monolayers, but they can be repurposed for the quantification of the size and shape of any other distinct cellular structure that can be stained (e.g., mitochondria or nuclei). Assessing these parameters is important in the characterization of the dynamic forces in adherent cell layer, including cell adhesion and actomyosin contractility that affects cell shape.

The article describes quantification of 1) the size and number of focal adhesions and 2) cell shape index and its distribution from confocal images of the confluent monolayers of MCF7 cells.

The methods presented here quantify some parameters of confluent adherent cell monolayers from multiple appropriately stained confocal images: adhesion to ...

Cytoskeleton and Focal Adhesion Organization Assay: An Immunofluorescence-based Method to Study Cell Adhesion and Spreading on Substrates

Source: Ali, M. Y et al., <a href="https://www.jove.com/v/52532"> Isolation of Primary Human Colon Tumor Cells from Surgical Tissues and Culturing Them Directly on Soft Elastic Substrates for Traction Cytometry.</a> J. Vis. Exp. (2015)
This video describes the technique of studying the cytoskeleton and focal adhesions in primary human colon cancer cells to understand its variations depending on substrate rigidity. This culture of cancer cells on soft and hard substrates allows various downstream biophysical measurements for cancer prognosis.

Source: Ali, M. Y et al.,  Isolation of Primary Human Colon Tumor Cells from Surgical Tissues and Culturing Them Directly on Soft Elastic Substrates for ...

Imaging Molecular Adhesion in Cell Rolling by Adhesion Footprint Assay

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