β Integrin-mediated Cell Adhesion Array Kit, fluorimetric

β Integrin-mediated Cell Adhesion Array Kit, fluorimetric

Chemicon (Millipore)

1 plate

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

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.

Human

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.

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.

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.

• 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

Chemicon

• P05556
• P05106
• P05107
• P16144
• P18564
• P06756
• P08648

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.

Fluorescent

96 wells

• ITGB1
• GPIIA
• VLAB
• OTTHUMP00000063732
• MDF2
• FNRB
• OTTHUMP00000063731
• OTTHUMP00000046253
• CD29
• OTTHUMP00000063733
• MSK12

Integrins, ECM, & Adhesion

β Integrin-mediated Cell Adhesion Array Kit, fluorimetric

• NM_002211.2
• NM_033666.1
• NM_033667.1
• NM_033668.1
• NM_033669.1
• NM_133376.1

· 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