Beta Integrin-Mediated Cell Adhesion Array Kit (Fluorimetric)

Detection Methods
Fluorescent

Detection Methods

Fluorescent

Description
Catalogue Number	ECM534
Brand Family	Chemicon®
Trade Name	Chemicon
Description	β Integrin-mediated Cell Adhesion Array Kit, fluorimetric
Overview	Introduction Cell adhesion plays a major role in cellular communication and regulation, and is of fundamental importance in the development and maintenance of tissues. A family of cell surface receptors, the integrins, mediates cell attachment to proteins of the extracellular matrix. Integrins are heterodimer molecules containing an alpha and beta transmembrane glycoprotein subunit that are non-covalently bound together (1-2). Historically, antibodies have been used to determine the integrin profiles of cells by immunoprecipitation, immunofluorescence, immunoblotting, and flow cytometry. These methods are time-consuming, laborious or require sophisticated equipment. The CHEMICON® Beta Integrin-Mediated Cell Adhesion Array kit is designed as a cost effective, efficient method for identification of cell surface integrins. The kit format uses 12 x 8-well removable strips in a plate frame for convenience and flexibility in designing assays. The Beta Integrin-Mediated Cell Adhesion Array can be completed within 1-2 hours, providing a rapid, quantitative assay for the characterization of beta integrin surface expression. In addition, Chemicon continues to provide numerous migration, invasion, and adhesion products including: · Alpha Integrin-Mediated Cell Adhesion Array Kit (Fluorimetric) (ECM533) · Alpha/Beta Integrin-Mediated Cell Adhesion Combo Array Kit (Fluorimetric) (ECM535) · Alpha Integrin-Mediated Cell Adhesion Array Kit (Colorimetric) (ECM530) · Beta Integrin-Mediated Cell Adhesion Array Kit (Colorimetric) (ECM531) · Alpha/Beta Integrin-Mediated Cell Adhesion Array Combo Kit (Colorimetric) (ECM532) · QCM™ 8μm 96-well Chemotaxis Cell Migration Assay (ECM510) · QCM™ 5μm 96-well Chemotaxis Cell Migration Assay (ECM512) · QCM™ 3μm 96-well Chemotaxis Cell Migration Assay (ECM515) · QCM™ 96-well Cell Invasion Assay (ECM555) · QCM™ 96-well Collagen-Based Cell Invasion Assay (ECM 556) · 24-well Insert Cell Migration and Invasion assay systems · CytoMatrix™ Cell Adhesion strips (ECM protein coated) · QuantiMatrix™ ECM protein ELISA Kits Test Principle The CHEMICON® Beta Integrin-Mediated Cell Adhesion Array Kit uses mouse monoclonal antibodies generated against human beta integrins/subunits (Ãeta1, Ãeta2, Ãeta3, Ãeta4, Ãeta6, alphaVÃeta5, and alpha5Ãeta1), that are immobilized onto a goat anti-mouse antibody coated microtiter plate. The plate is then used to capture cells expressing these integrins on their cell surface; goat anti-mouse antibody coated wells are provided as a negative assay control. Each strip (8 wells total) consists of 7 integrin antibody coated wells and one negative control well (see plate layout on page 3). Experimental cells are seeded onto the coated substrate and incubated. Subsequently, unbound cells are washed away, and the adherent cells are lysed and detected by the patented CyQuant GR dye (Molecular Probes) (3-4). This green-fluorescent dye exhibits strong fluorescence enhancement when bound to cellular nucleic acids (5). Relative cell attachment is determined using a fluorescence plate reader. Additional information about the monoclonals provided in the kit: Monoclonal / Known Species Reactivity / Clone Number Mouse anti-Beta1 [CD29] / Hu, Mky, Rb / P4G11 Mouse anti-Beta2 [CD18] / Hu / P4H9 Mouse anti-Beta3 [CD61] / Hu / 25E11 Mouse anti-Beta4 [CD104] / Hu / ASC-9 Mouse anti-Beta6 / Hu, Rt, Mky, Rb / CSb6 Mouse anti-AlphaV/ Beta5 [CD51/CD61] / Hu, Chk / P1F6 Mouse anti-Alpha5/ Beta1 [VLA5, FNR] / Hu, Por / HA5
Materials Required but Not Delivered	· Multi-channel and/or repeating pipettes · Harvesting buffer: EDTA or trypsin cell detachment buffer. Suggested formulations include a) 2 mM EDTA/PBS, b) 0.05% trypsin in Hanks Balanced Salt Solution (HBSS) containing 25 mM HEPES, or other cell detachment formulations as optimized by individual investigators. Note: Trypsin cell detachment buffer maybe required for difficult cell lines. Allow sufficient time for cell receptor recovery. · Tissue culture growth medium appropriate for subject cells, such as DMEM containing 10% FBS · Quenching Medium: serum-free medium, such as DMEM, EMEM, or FBM (fibroblast basal media), containing 5% BSA Note: Quenching Medium must contain divalent cations (Mg2+, Ca2+) sufficient for quenching EDTA in the harvesting buffer. · Sterile PBS or HBSS to wash cells · Deionized water · Low speed centrifuge and tubes for cell harvesting · CO2 incubator appropriate for subject cells · Sterile cell culture hood · Hemacytometer and microscope for counting cells · Trypan Blue or equivalent viability stain · Fluorescence plate reader with 485nm and 530nm filter set · 96-well plate suitable for fluorescence measurement

Description

Catalogue Number

ECM534

Brand Family

Chemicon®

Trade Name

Chemicon

Description

β Integrin-mediated Cell Adhesion Array Kit, fluorimetric

Overview

Introduction

Cell adhesion plays a major role in cellular communication and regulation, and is of fundamental importance in the development and maintenance of tissues. A family of cell surface receptors, the integrins, mediates cell attachment to proteins of the extracellular matrix. Integrins are heterodimer molecules containing an alpha and beta transmembrane glycoprotein subunit that are non-covalently bound together (1-2). Historically, antibodies have been used to determine the integrin profiles of cells by immunoprecipitation, immunofluorescence, immunoblotting, and flow cytometry. These methods are time-consuming, laborious or require sophisticated equipment.

The CHEMICON® Beta Integrin-Mediated Cell Adhesion Array kit is designed as a cost effective, efficient method for identification of cell surface integrins. The kit format uses 12 x 8-well removable strips in a plate frame for convenience and flexibility in designing assays. The Beta Integrin-Mediated Cell Adhesion Array can be completed within 1-2 hours, providing a rapid, quantitative assay for the characterization of beta integrin surface expression.

In addition, Chemicon continues to provide numerous migration, invasion, and adhesion products including:

· Alpha Integrin-Mediated Cell Adhesion Array Kit (Fluorimetric) (ECM533)

· Alpha/Beta Integrin-Mediated Cell Adhesion Combo Array Kit (Fluorimetric) (ECM535)

· Alpha Integrin-Mediated Cell Adhesion Array Kit (Colorimetric) (ECM530)

· Beta Integrin-Mediated Cell Adhesion Array Kit (Colorimetric) (ECM531)

· Alpha/Beta Integrin-Mediated Cell Adhesion Array Combo Kit (Colorimetric) (ECM532)

· QCM™ 8μm 96-well Chemotaxis Cell Migration Assay (ECM510)

· QCM™ 5μm 96-well Chemotaxis Cell Migration Assay (ECM512)

· QCM™ 3μm 96-well Chemotaxis Cell Migration Assay (ECM515)

· QCM™ 96-well Cell Invasion Assay (ECM555)

· QCM™ 96-well Collagen-Based Cell Invasion Assay (ECM 556)

· 24-well Insert Cell Migration and Invasion assay systems

· CytoMatrix™ Cell Adhesion strips (ECM protein coated)

· QuantiMatrix™ ECM protein ELISA Kits

Test Principle

The CHEMICON® Beta Integrin-Mediated Cell Adhesion Array Kit uses mouse monoclonal antibodies generated against human beta integrins/subunits (Ãeta1, Ãeta2, Ãeta3, Ãeta4, Ãeta6, alphaVÃeta5, and alpha5Ãeta1), that are immobilized onto a goat anti-mouse antibody coated microtiter plate. The plate is then used to capture cells expressing these integrins on their cell surface; goat anti-mouse antibody coated wells are provided as a negative assay control. Each strip (8 wells total) consists of 7 integrin antibody coated wells and one negative control well (see plate layout on page 3). Experimental cells are seeded onto the coated substrate and incubated. Subsequently, unbound cells are washed away, and the adherent cells are lysed and detected by the patented CyQuant GR dye (Molecular Probes) (3-4). This green-fluorescent dye exhibits strong fluorescence enhancement when bound to cellular nucleic acids (5). Relative cell attachment is determined using a fluorescence plate reader.

Additional information about the monoclonals provided in the kit:

Monoclonal / Known Species Reactivity / Clone Number

Mouse anti-Beta1 [CD29] / Hu, Mky, Rb / P4G11

Mouse anti-Beta2 [CD18] / Hu / P4H9

Mouse anti-Beta3 [CD61] / Hu / 25E11

Mouse anti-Beta4 [CD104] / Hu / ASC-9

Mouse anti-Beta6 / Hu, Rt, Mky, Rb / CSb6

Mouse anti-AlphaV/ Beta5 [CD51/CD61] / Hu, Chk / P1F6

Mouse anti-Alpha5/ Beta1 [VLA5, FNR] / Hu, Por / HA5

Materials Required but Not Delivered

· Multi-channel and/or repeating pipettes

· Harvesting buffer: EDTA or trypsin cell detachment buffer. Suggested formulations include a) 2 mM EDTA/PBS, b) 0.05% trypsin in Hanks Balanced Salt Solution (HBSS) containing 25 mM HEPES, or other cell detachment formulations as optimized by individual investigators.

Note: Trypsin cell detachment buffer maybe required for difficult cell lines. Allow sufficient time for cell receptor recovery.

· Tissue culture growth medium appropriate for subject cells, such as DMEM containing 10% FBS

· Quenching Medium: serum-free medium, such as DMEM, EMEM, or FBM (fibroblast basal media), containing 5% BSA

Note: Quenching Medium must contain divalent cations (Mg2+, Ca2+) sufficient for quenching EDTA in the harvesting buffer.

· Sterile PBS or HBSS to wash cells

· Deionized water

· Low speed centrifuge and tubes for cell harvesting

· CO2 incubator appropriate for subject cells

· Sterile cell culture hood

· Hemacytometer and microscope for counting cells

· Trypan Blue or equivalent viability stain

· Fluorescence plate reader with 485nm and 530nm filter set

· 96-well plate suitable for fluorescence measurement

Product Information
Components	Beta Integrin Array Plate: (PN: 90600) One 96-well plate with 12 strips. Each strip contains one well each of the mouse anti-beta integrin monoclonal antibodies: (beta1, beta2, beta3, beta4, beta6, alphaVbeta5, and alpha5beta1), and one goat anti-mouse negative well. See the plate layout on insert data sheet. 4X Cell Lysis Buffer: (Part No. 90130) One bottle - 16 mL CyQuant GR Dye®: (Part No. 90132) One vial - 75 μL Assay Buffer: (Part No. 90601) One bottle - 100 mL
Detection method	Fluorescent

Product Information

Components

Beta Integrin Array Plate: (PN: 90600) One 96-well plate with 12 strips. Each strip contains one well each of the mouse anti-beta integrin monoclonal antibodies: (beta1, beta2, beta3, beta4, beta6, alphaVbeta5, and alpha5beta1), and one goat anti-mouse negative well. See the plate layout on insert data sheet.
4X Cell Lysis Buffer: (Part No. 90130) One bottle - 16 mL
CyQuant GR Dye®: (Part No. 90132) One vial - 75 μL
Assay Buffer: (Part No. 90601) One bottle - 100 mL

Detection method

Fluorescent

Applications
Application	The Beta Integrin-Mediated Cell Adhesion Array Kit can be used for assessing the presence or absence of specific integrins on the cell surface.
Application Notes	The CHEMICON® Beta Integrin-Mediated Cell Adhesion Array Kit can be used for assessing the presence or absence of specific integrins on the cell surface. This assay can substitute for FACS analysis (6) and is useful for screening human adherent cells for cell surface integrins or for monitoring in vitro cell differentiation or genetic modification of cells. For Research Use Only. Not for use in diagnostic procedures.

Applications

Application

The Beta Integrin-Mediated Cell Adhesion Array Kit can be used for assessing the presence or absence of specific integrins on the cell surface.

Application Notes

The CHEMICON® Beta Integrin-Mediated Cell Adhesion Array Kit can be used for assessing the presence or absence of specific integrins on the cell surface. This assay can substitute for FACS analysis (6) and is useful for screening human adherent cells for cell surface integrins or for monitoring in vitro cell differentiation or genetic modification of cells.

For Research Use Only. Not for use in diagnostic procedures.

Biological Information
Species Reactivity	Human
Entrez Gene Number	NM_002211.2 NM_033666.1 NM_033667.1 NM_033668.1 NM_033669.1 NM_133376.1
Entrez Gene Summary	Integrins are heterodimers comprised of alpha and beta subunits, that are noncovalently associated transmembrane glycoprotein receptors. Different combinations of alpha and beta polypeptides form complexes that vary in their ligand-binding specificities. Integrins mediate cell-matrix or cell-cell adhesion, and transduced signals that regulate gene expression and cell growth. This gene encodes the integrin beta 4 subunit, a receptor for the laminins. This subunit tends to associate with alpha 6 subunit and is likely to play a pivotal role in the biology of invasive carcinoma. Mutations in this gene are associated with epidermolysis bullosa with pyloric atresia. Multiple alternatively spliced transcript variants encoding distinct isoforms have been found for this gene.
Gene Symbol	ITGB1 GPIIA VLAB OTTHUMP00000063732 MDF2 FNRB OTTHUMP00000063731 OTTHUMP00000046253 CD29 OTTHUMP00000063733 MSK12
UniProt Number	P05556 P05106 P05107 P16144 P18564 P06756 P08648
UniProt Summary	FUNCTION: SwissProt: P18564 # Integrin alpha-V/beta-6 is a receptor for fibronectin and cytotactin. It recognizes the sequence R-G-D in its ligands. SIZE: 788 amino acids; 85936 Da SUBUNIT: Heterodimer of an alpha and a beta subunit. Beta-6 associates with alpha-V. Interacts with FLNB. SUBCELLULAR LOCATION: Membrane; Single-pass type I membrane protein. SIMILARITY: SwissProt: P18564 ## Belongs to the integrin beta chain family. & Contains 1 VWFA domain.

Biological Information

Species Reactivity

Human

Entrez Gene Number

Entrez Gene Summary

Integrins are heterodimers comprised of alpha and beta subunits, that are noncovalently associated transmembrane glycoprotein receptors. Different combinations of alpha and beta polypeptides form complexes that vary in their ligand-binding specificities. Integrins mediate cell-matrix or cell-cell adhesion, and transduced signals that regulate gene expression and cell growth. This gene encodes the integrin beta 4 subunit, a receptor for the laminins. This subunit tends to associate with alpha 6 subunit and is likely to play a pivotal role in the biology of invasive carcinoma. Mutations in this gene are associated with epidermolysis bullosa with pyloric atresia. Multiple alternatively spliced transcript variants encoding distinct isoforms have been found for this gene.

Gene Symbol

ITGB1
GPIIA
VLAB
OTTHUMP00000063732
MDF2
FNRB
OTTHUMP00000063731
OTTHUMP00000046253
CD29
OTTHUMP00000063733
MSK12

UniProt Number

UniProt Summary

FUNCTION: SwissProt: P18564 # Integrin alpha-V/beta-6 is a receptor for fibronectin and cytotactin. It recognizes the sequence R-G-D in its ligands.
SIZE: 788 amino acids; 85936 Da
SUBUNIT: Heterodimer of an alpha and a beta subunit. Beta-6 associates with alpha-V. Interacts with FLNB.
SUBCELLULAR LOCATION: Membrane; Single-pass type I membrane protein.
SIMILARITY: SwissProt: P18564 ## Belongs to the integrin beta chain family. & Contains 1 VWFA domain.

Product Usage Statements
Usage Statement	Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.

Product Usage Statements

Usage Statement

Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.

Storage and Shipping Information
Storage Conditions	The Beta Integrin Array Plate can be stored at 2° to 8°C in the foil pouch up to its expiration date. Unused strips may be placed back in the pouch for storage. Ensure that the desiccant remains in the pouch, and that the pouch is securely closed. Keep the remaining kit components at 2° to 8°C. Precautions No data is available on the biological toxicity of CyQuant GR® dye. This reagent binds to nucleic acids, and as such, should be treated as a potential mutagen which may cause cancer and heritable genetic damage. Handle with caution. The DMSO stock solution should be handled with special caution as DMSO can facilitate the entry of organic molecules into tissues.

Storage and Shipping Information

Storage Conditions

The Beta Integrin Array Plate can be stored at 2° to 8°C in the foil pouch up to its expiration date. Unused strips may be placed back in the pouch for storage. Ensure that the desiccant remains in the pouch, and that the pouch is securely closed. Keep the remaining kit components at 2° to 8°C.

Precautions

No data is available on the biological toxicity of CyQuant GR® dye. This reagent binds to nucleic acids, and as such, should be treated as a potential mutagen which may cause cancer and heritable genetic damage. Handle with caution. The DMSO stock solution should be handled with special caution as DMSO can facilitate the entry of organic molecules into tissues.

Packaging Information
Material Size	1 plate
Material Package	96 wells

Packaging Information

Material Size

1 plate

Material Package

96 wells

实验耗材

分子生物学试剂

蛋白与免疫学

生化试剂

细胞及检测试剂

色谱及层析

仪器设备

实验服务

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Beta Integrin-Mediated Cell Adhesion Array Kit (Fluorimetric)

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