Specthophotmetric quantification of cell-cell adherence by an enzyme-linked immuno-cell adhesion assay

Collagen, a major structural protein of the ECM, is known for its high cell adherence capacity. This study was conducted to identify regions in collagen that harbour such bioactivity. Collagen from tendon was hydrolysed and the peptides fractionated using ion-exchange chromatography (IEC). Isolated peptide fractions were coated onto disposable dishes and screened for cell adherence and proliferative abilities. Active IEC fractions were further purified by chromatography, and two peptides, C2 and E1 with cell adhesion ability, were isolated. A cell adhesion assay done with different amounts of C2 coated onto disposable dishes revealed the maximum adhesion to be 94.6%, compared with 80% for collagen coated dishes and an optimum peptide coating density of 0.507 nmoles per cm2 area of the dish. Growth of cells on C2, collagen, and E1 revealed a similar pattern and a reduction in the doubling time compared with cells grown on uncoated dishes. C2 had a mass of 2.046 kDa with 22 residues, and sequence analysis revealed a higher percentage occurrence of hydrophilic residues compared with other regions in collagen. Docking studies revealed GDDGEA in C2 as the probable site of interaction with integrins α2β1 and α1β1, and stability studies proved C2 to be mostly protease-resistant.

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Changes in E-cadherin rigidity sensing regulate cell adhesion

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1618676114 ◽

2017 ◽

Vol 114 (29) ◽

pp. E5835-E5844 ◽

Cited By ~ 34

Author(s):

Caitlin Collins ◽

Aleksandra K. Denisin ◽

Beth L. Pruitt ◽

W. James Nelson

Keyword(s):

Cell Adhesion ◽

Signaling Molecules ◽

Membrane Dynamics ◽

Cell Contact ◽

Adhesion Assay ◽

Cell Periphery ◽

Actin Organization ◽

Rigidity Sensing ◽

E Cadherin ◽

Cell Cell

Mechanical cues are sensed and transduced by cell adhesion complexes to regulate diverse cell behaviors. Extracellular matrix (ECM) rigidity sensing by integrin adhesions has been well studied, but rigidity sensing by cadherins during cell adhesion is largely unexplored. Using mechanically tunable polyacrylamide (PA) gels functionalized with the extracellular domain of E-cadherin (Ecad-Fc), we showed that E-cadherin–dependent epithelial cell adhesion was sensitive to changes in PA gel elastic modulus that produced striking differences in cell morphology, actin organization, and membrane dynamics. Traction force microscopy (TFM) revealed that cells produced the greatest tractions at the cell periphery, where distinct types of actin-based membrane protrusions formed. Cells responded to substrate rigidity by reorganizing the distribution and size of high-traction-stress regions at the cell periphery. Differences in adhesion and protrusion dynamics were mediated by balancing the activities of specific signaling molecules. Cell adhesion to a 30-kPa Ecad-Fc PA gel required Cdc42- and formin-dependent filopodia formation, whereas adhesion to a 60-kPa Ecad-Fc PA gel induced Arp2/3-dependent lamellipodial protrusions. A quantitative 3D cell–cell adhesion assay and live cell imaging of cell–cell contact formation revealed that inhibition of Cdc42, formin, and Arp2/3 activities blocked the initiation, but not the maintenance of established cell–cell adhesions. These results indicate that the same signaling molecules activated by E-cadherin rigidity sensing on PA gels contribute to actin organization and membrane dynamics during cell–cell adhesion. We hypothesize that a transition in the stiffness of E-cadherin homotypic interactions regulates actin and membrane dynamics during initial stages of cell–cell adhesion.

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Dissection of Organizer and Animal Pole Explants from Xenopus laevis Embryos and Assembly of a Cell Adhesion Assay

Journal of Visualized Experiments ◽

10.3791/187 ◽

2007 ◽

Cited By ~ 1

Author(s):

Souichi Ogata ◽

Ken W.Y. Cho

Keyword(s):

Cell Adhesion ◽

Xenopus Laevis ◽

Adhesion Assay ◽

Animal Pole ◽

Cell Adhesion Assay ◽

A Cell ◽

Xenopus Laevis Embryos

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A centrifugation cell adhesion assay for high-throughput screening of biomaterial surfaces

Journal of Biomedical Materials Research Part A ◽

10.1002/jbm.a.10122 ◽

2003 ◽

Vol 67A (1) ◽

pp. 328-333 ◽

Cited By ~ 123

Author(s):

Catherine D. Reyes ◽

Andrés J. García

Keyword(s):

Cell Adhesion ◽

High Throughput ◽

High Throughput Screening ◽

Adhesion Assay ◽

Cell Adhesion Assay ◽

Biomaterial Surfaces

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Development of a high throughput Pseudomonas aeruginosa epithelial cell adhesion assay

Journal of Microbiological Methods ◽

10.1016/s0167-7012(02)00193-8 ◽

2003 ◽

Vol 52 (3) ◽

pp. 361-366 ◽

Cited By ~ 8

Author(s):

Britta Swanson ◽

Richard Savel ◽

Frank Szoka ◽

Teiji Sawa ◽

Jeanine Wiener-Kronish

Keyword(s):

Pseudomonas Aeruginosa ◽

Cell Adhesion ◽

Epithelial Cell ◽

High Throughput ◽

Adhesion Assay ◽

Cell Adhesion Assay

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Neutrophils Rolling on Immobilised Platelets Migrate into Homotypic Aggregates after Activation

Thrombosis and Haemostasis ◽

10.1055/s-0037-1615037 ◽

1998 ◽

Vol 79 (06) ◽

pp. 1177-1183 ◽

Cited By ~ 14

Author(s):

Christopher Buckley ◽

David Simmons ◽

Gerard Nash ◽

G. E. Rainger

Keyword(s):

Cell Adhesion ◽

Vascular Disease ◽

Adhesion Molecules ◽

Platelet Activating Factor ◽

Vascular Occlusion ◽

Adhesion Assay ◽

Αvβ3 Integrin ◽

Projected Area ◽

Activated Platelets ◽

Cell Cell

SummaryInteractions between platelets and leucocytes are implicated in the pathology of thrombotic vascular disease. Using a flow-based adhesion assay we have investigated a novel route for the formation of neutrophil aggregates on the surface of immobilised activated platelets. Neutrophils perfused over a platelet monolayer formed numerous rolling attachments but rapidly stopped and spread after the superfusion of N-formyl-methionyl-leucyl-phenylalanine or platelet-activating factor (both at 10–7 M). Subsequent integrin-mediated migration across the platelet monolayer enabled formation of homotypic neutrophil aggregates, which was significant within 2.5 min of receipt of either stimulus. Aggregates increased in size with time and had an average projected area of ~500 μm2 after 10 min. Increasing size was correlated with an increasing tendency for movement downstream and large aggregates sometimes tumbled in that direction. The formation and stability of homotypic aggregates was dependent on several adhesive mechanisms. Antibody blockade demonstrated that interactions involving CD11a/ CD18 and ICAM-3, between αvβ3-integrin and CD31 and between L-selectin and an unidentified counter-ligand were all required for the complete aggregatory response. Furthermore, blockade of L-selectin allowed initial aggregation which then reversed, suggesting that this receptor might regulate the interactions between other adhesion molecules that directly supported cell-cell adhesion. We propose that this novel route for leucocyte aggregation could promote vascular occlusion in thrombotic vessels or at distal sites in the event of embolisation.

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