α Integrin-mediated Cell Adhesion Array Kit, fluorimetric

α 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® Alpha 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 Alpha Integrin-Mediated Cell Adhesion Array can be completed within 1-2 hours, providing a rapid, quantitative assay for the characterization of alpha integrin surface expression.

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

· Beta Integrin-Mediated Cell Adhesion Array Kit (Fluorimetric) (ECM534)

· 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® Alpha Integrin-Mediated Cell Adhesion Array Kit uses mouse monoclonal antibodies generated against human alpha integrins/subunits (alpha1, alpha2, alpha3, alpha4, alpha5, alphaV, and alphavbeta3), 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-Alpha1 [CD49a] / Hu / FB12

Mouse anti-Alpha2 [CD49b] / Hu / P1E6

Mouse anti-Alpha3 [CD49c] / Hu / P1B5

Mouse anti-Alpha4 [CD49d] / Hu, Prim / P1H4

Mouse anti-Alpha5 [CD49e] / Hu / P1D6

Mouse anti-AlphaV [CD51] / Hu / P3G8

Mouse anti-AlphaV/Beta3 [CD51/CD61] / Hu, Bov, Por, Av, Can, Chk, Mky, Rb / LM609

The CHEMICON® Alpha 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.

This gene encodes the alpha 1 subunit of integrin receptors. This protein heterodimerizes with the beta 1 subunit to form a cell-surface receptor for collagen and laminin. The heterodimeric receptor is involved in cell-cell adhesion and may play a role in inflammation and fibrosis. The alpha 1 subunit contains an inserted (I) von Willebrand factor type I domain which is thought to be involved in collagen binding.

FUNCTION: SwissProt: P56199 # Integrin alpha-1/beta-1 is a receptor for laminin and collagen. It recognizes the proline-hydroxylated sequence G-F-P-G- E-R in collagen.
SIZE: 1179 amino acids; 130848 Da
SUBUNIT: Heterodimer of an alpha and a beta subunit. Alpha-1 associates with beta-1.
SUBCELLULAR LOCATION: Membrane; Single-pass type I membrane protein.
DOMAIN: SwissProt: P56199 The integrin I-domain (insert) is a VWFA domain. Integrins with I-domains do not undergo protease cleavage.
SIMILARITY: Belongs to the integrin alpha chain family. & Contains 7 FG-GAP repeats. & Contains 1 VWFA domain.

• Alpha Integrin Array Plate: (PN: 90599) One 96-well plate with 12 strips. Each strip contains one well each of the mouse anti-alpha integrin monoclonal antibodies: (alpha1, alpha2, alpha3, alpha4, alpha5, alphaV, alphavbeta3), 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

• P56199
• P17301
• P26006
• P13612
• P08648
• P06756

The Alpha 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

• ITGA1
• CD49a
• VLA1
• VLA-1

Integrins, ECM, & Adhesion

α Integrin-mediated Cell Adhesion Array Kit, fluorimetric

NM_181501.1

· 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% BS

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