SRU Biosystems - Cell-Based Applications >> Cell Adhesion

Cell-Based Applications >> Cell Adhesion

Cell adhesion molecules such as integrins, cadherins and selectins as well as adhesion-linked signaling molecules are important drug targets in various types of disease including cancer, infection and inflammation. Cell-matrix binding assays have been difficult to develop and deploy in screening formats. The standard cell adhesion assays, such as ELISA-like methods, require multiple steps, labeling of the cells and repeat washing of the cells to determine specifically bound cell numbers. These requirements contribute to poor reproducibility and hinder assay throughput. BIND® technology provides for robust, label-free cell adhesion assays that are easily applied to both high throughput screening and compound profiling.

BIND can be used in an array of formats for cellular adhesion assays. Biosensors can be coated with any number of extracellular matrix proteins such as fibronectin, collagen or laminin, or with another target of interest. Alternatively, the biosensor can be coated with antibodies enabling measurement of cell adhesion specificity. Typical adhesion assays can be run in 3 easy steps: coating of biosensor plates, addition & stimulation of cells and measurement of PWV on a BIND Reader as a readout of the level of adhesion. A positive PWV shift indicates a tighter association with the biosensor, and a negative PWV shift indicates a looser attachment. No labeling or washing is required simplifying assay development and increasing throughput capabilities.

Application Features

Label-free, Live Cell Assay
Low to High Throughput Formats
Capable of Measuring Adhesion in Real-time
Biosensor Surface Coating Flexibility

Biosensor Surface Flexibility

The flexibility of the BIND system allows for focused exploration of specific adhesion pathways even in an ultra-high throughput 1536-well format. Biosensors can be coated with ECM proteins, antibodies, peptides, or any protein of interest and cellular adhesion measured. The BIND Reader software interface shown in Figure 1 depicts the results of an assay testing the adhesion of two different cell lines overexpressing muscarinic receptors to specific extracellular matrix proteins. This easy-to-read software display, called the heat map, shows the change in PWV for each individual well. Red indicates the largest PWV shift while blue indicates the smallest PWV shift. These data clearly show the differences in adhesion of the same cell line to multiple ECM proteins as well as the differences in adhesion of specific cell lines expressing different receptors to the same ECM protein.

1536-well Adhesion Assay using An Array of ECM Proteins

Figure 1 – A 1536-well BIND Biosensor was coated with BSA or one of 3 extracellular matrix proteins (fibronectin, FN; collagen, COLL; laminin, LAM). Cells overexpressing either the M4 or M5 muscarinic receptor (courtesy of Millipore) were incubated on BIND Biosensors and PWV measured. Results are displayed in heat map format with the largest PWV shifts depicted in red and the smallest PWV shifts depicted in blue.

Receptor Mediated Adhesion Specificity

BIND adhesion assays can be used to both quantify the level of cell adhesion and as a tool to examine the requirements, specificity and inhibition of adhesion. The integrin-dependence of cell binding to ECM proteins can be examined using a panel of antibodies against integrin subunits and measuring their affects on adhesion. Figure 2 summarizes results of adhesion assays in which THP-1 cells were plated on fibronectin-coated Biosensors in the presence and absence of anti-integrin mAbs to characterize the specific interaction between fibronectin and endogenously expressed α4β1 integrin. Three different anti-β1 mAbs lowered the level of cellular attachment to baseline levels (BSA with Mn++) while an anti-α5 mAb had no affects on adhesion.

Integrin Subunit Specificity

Figure 2 - THP-1 cells were plated on Fibronectin or BSA-coated Biosensors in the presence of Mg++ (±Mn++) and attachment measured using a BIND Reader. Cells were then incubated with and without various antibodies against specific integrin subunits and attachment measured to assess changes in adhesion.

Adhesion Inhibition

BIND adhesion assays can be used to screen compounds for inhibition of cell adhesion. Figure 3 depicts an assay used to screen compounds for inhibition of VLA-4 (Very Late Antigen-1 or α4β1) binding to VCAM (vascular cell adhesion molecule-1). VCAM-1 is an endothelial ligand for VLA-4 of the β1 subfamily of integrins and for integrin α4β7. VCAM-1 promotes the adhesion of lymphocytes, monocytes, eosinophils, and basophils. Interestingly, certain melanoma cells can use VCAM-1 to adhere to the endothelium and VCAM-1 may participate in monocyte recruitment to atherosclerotic sites. As a result, VCAM-1 is a potential target drug target.

Adhesion Inhibitor Identification

Figure 3 - BIND biosensors were coated with an antibody specific for VCAM. Recombinant VCAM was immobilized to the biosensor surface via the antibody. Cells were plated on coated biosensors and adhesion measured in the presence and absence of test compounds using a BIND Reader.

Cation Dependency

Jukat Cell Adhesion

BIND adhesion assays also allow for examination of the cation-dependency of adhesion. The addition of EDTA to Jurkat cells demonstrates a reversal of cell adhesion in Mn++ mediated adhesion (Figure 4).

Figure 4 – Jurkat cells were plated on fibronectin-coated BIND biosensors in the presence or absence of 1mM Mn++. In addition EDTA was added to some wells at time zero and some wells after 40 minutes.

CHO Cell Adhesion

The cation dependency of CHO-K1 adhesion to fibronectin was assessed using BIND. While CHO cells were able to adhere to fibronection-coated biosensors, adhesion was enhanced by the addition of several different cations.

Figure 5 – CHO-K1 cells were plated in Hanks Buffered Saline without calcium on fibronectin or BSA-coated BIND biosensors and PWV measured over approximately 6 hours. Either Ca++, Mg++ or Mn++ was added to cells and PWV measured for an additional 3 hours.

Contact Us
Home Company Technology Products Applications Resource Center


Applications Cell-Based Applications • GPCRs • Ligand-Gated Ion Channels • Cell Adhesion Biochemical Applications	Cell-Based Applications >> Cell Adhesion Cell adhesion molecules such as integrins, cadherins and selectins as well as adhesion-linked signaling molecules are important drug targets in various types of disease including cancer, infection and inflammation. Cell-matrix binding assays have been difficult to develop and deploy in screening formats. The standard cell adhesion assays, such as ELISA-like methods, require multiple steps, labeling of the cells and repeat washing of the cells to determine specifically bound cell numbers. These requirements contribute to poor reproducibility and hinder assay throughput. BIND® technology provides for robust, label-free cell adhesion assays that are easily applied to both high throughput screening and compound profiling. BIND can be used in an array of formats for cellular adhesion assays. Biosensors can be coated with any number of extracellular matrix proteins such as fibronectin, collagen or laminin, or with another target of interest. Alternatively, the biosensor can be coated with antibodies enabling measurement of cell adhesion specificity. Typical adhesion assays can be run in 3 easy steps: coating of biosensor plates, addition & stimulation of cells and measurement of PWV on a BIND Reader as a readout of the level of adhesion. A positive PWV shift indicates a tighter association with the biosensor, and a negative PWV shift indicates a looser attachment. No labeling or washing is required simplifying assay development and increasing throughput capabilities. Application Features Label-free, Live Cell Assay Low to High Throughput Formats Capable of Measuring Adhesion in Real-time Biosensor Surface Coating Flexibility Biosensor Surface Flexibility The flexibility of the BIND system allows for focused exploration of specific adhesion pathways even in an ultra-high throughput 1536-well format. Biosensors can be coated with ECM proteins, antibodies, peptides, or any protein of interest and cellular adhesion measured. The BIND Reader software interface shown in Figure 1 depicts the results of an assay testing the adhesion of two different cell lines overexpressing muscarinic receptors to specific extracellular matrix proteins. This easy-to-read software display, called the heat map, shows the change in PWV for each individual well. Red indicates the largest PWV shift while blue indicates the smallest PWV shift. These data clearly show the differences in adhesion of the same cell line to multiple ECM proteins as well as the differences in adhesion of specific cell lines expressing different receptors to the same ECM protein. 1536-well Adhesion Assay using An Array of ECM Proteins Figure 1 – A 1536-well BIND Biosensor was coated with BSA or one of 3 extracellular matrix proteins (fibronectin, FN; collagen, COLL; laminin, LAM). Cells overexpressing either the M4 or M5 muscarinic receptor (courtesy of Millipore) were incubated on BIND Biosensors and PWV measured. Results are displayed in heat map format with the largest PWV shifts depicted in red and the smallest PWV shifts depicted in blue. Receptor Mediated Adhesion Specificity BIND adhesion assays can be used to both quantify the level of cell adhesion and as a tool to examine the requirements, specificity and inhibition of adhesion. The integrin-dependence of cell binding to ECM proteins can be examined using a panel of antibodies against integrin subunits and measuring their affects on adhesion. Figure 2 summarizes results of adhesion assays in which THP-1 cells were plated on fibronectin-coated Biosensors in the presence and absence of anti-integrin mAbs to characterize the specific interaction between fibronectin and endogenously expressed α4β1 integrin. Three different anti-β1 mAbs lowered the level of cellular attachment to baseline levels (BSA with Mn++) while an anti-α5 mAb had no affects on adhesion. Integrin Subunit Specificity Figure 2 - THP-1 cells were plated on Fibronectin or BSA-coated Biosensors in the presence of Mg++ (±Mn++) and attachment measured using a BIND Reader. Cells were then incubated with and without various antibodies against specific integrin subunits and attachment measured to assess changes in adhesion. Adhesion Inhibition BIND adhesion assays can be used to screen compounds for inhibition of cell adhesion. Figure 3 depicts an assay used to screen compounds for inhibition of VLA-4 (Very Late Antigen-1 or α4β1) binding to VCAM (vascular cell adhesion molecule-1). VCAM-1 is an endothelial ligand for VLA-4 of the β1 subfamily of integrins and for integrin α4β7. VCAM-1 promotes the adhesion of lymphocytes, monocytes, eosinophils, and basophils. Interestingly, certain melanoma cells can use VCAM-1 to adhere to the endothelium and VCAM-1 may participate in monocyte recruitment to atherosclerotic sites. As a result, VCAM-1 is a potential target drug target. Adhesion Inhibitor Identification Figure 3 - BIND biosensors were coated with an antibody specific for VCAM. Recombinant VCAM was immobilized to the biosensor surface via the antibody. Cells were plated on coated biosensors and adhesion measured in the presence and absence of test compounds using a BIND Reader. Cation Dependency Jukat Cell Adhesion BIND adhesion assays also allow for examination of the cation-dependency of adhesion. The addition of EDTA to Jurkat cells demonstrates a reversal of cell adhesion in Mn++ mediated adhesion (Figure 4). Figure 4 – Jurkat cells were plated on fibronectin-coated BIND biosensors in the presence or absence of 1mM Mn++. In addition EDTA was added to some wells at time zero and some wells after 40 minutes. CHO Cell Adhesion The cation dependency of CHO-K1 adhesion to fibronectin was assessed using BIND. While CHO cells were able to adhere to fibronection-coated biosensors, adhesion was enhanced by the addition of several different cations. Figure 5 – CHO-K1 cells were plated in Hanks Buffered Saline without calcium on fibronectin or BSA-coated BIND biosensors and PWV measured over approximately 6 hours. Either Ca++, Mg++ or Mn++ was added to cells and PWV measured for an additional 3 hours.
Company \| Technology \| Products \| Applications \| Resource Center \| Contact Us © 2000-2010 SRU Biosystems, Inc.