ECM671 QCM™ Gelatin Invadopodia Assay (Red)

ECM671
32 assays (1 kit)  Enough Reagents for 4 x 8-well Chamber Slides (32 assays)
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      Overview

      Replacement Information

      Key Specifications Table

      Key ApplicationsDetection Methods
      Invasion Assay, Migration Fluorescent
      Description
      Catalogue NumberECM671
      Brand Family Chemicon®
      Trade Name
      • QCM
      • Chemicon
      DescriptionQCM™ Gelatin Invadopodia Assay (Red)
      OverviewRead our application note in Nature Methods!
      http://www.nature.com/app_notes/nmeth/2012/121007/pdf/an8622.pdf
      (Click Here!)

      Also available: Cell Comb™ Scratch Assay! Get biochemical data from a scratch assay! Click Here

      Invasion of cells through layers of extracellular matrix is a key step in tumor metastasis, inflammation, and development. The process of invasion involves several stages, including adhesion to the matrix, degradation of proximal matrix molecules, extension and traction of the cell on the newly revealed matrix, and movement of the cell body through the resulting gap in the matrix (Friedl and Wolf, 2009). Each of these stages of invasion is executed by a suite of proteins, including proteases, integrins, GTPases, kinases, and cytoskeleton-interacting proteins.

      Classical methods for analyzing cellular invasion involve application of cells to one side of a layer of gelled matrix molecules and quantifying the relative number of cells that have traversed the layer. Such methods are extremely useful for analyzing invasion at the cell population level, but analysis of the subcellular events mediating the stages of invasion require techniques with higher resolution. The method that has been most informative for pinpointing regions of the cell that initiate invasion involve plating cells on a culture surface coated with a thin layer of fluorescently labeled matrix, and visualizing regions where the cell has degraded the matrix to create an area devoid of fluorescence (Chen et al., 1984). Such assays have revealed that invasive cells extend small localized protrusions that preferentially degrade the matrix. These protrusions are termed invadopodia in cancerous cells, and podosomes in non-malignant cells such as macrophages (Ayala et al., 2006). Many molecules orchestrate the formation and function of invadopodia; a few of the key molecular events include Src phosphorylation of scaffolding protein Tks5 (Seals et al., 2005), N-WASP activation and cortactin regulation of the Arp2/3 complex to induce actin polymerization (Yamaguchi et al., 2005; Weaver, 2006), and generation of reactive oxygen species by NADPH oxidases (Diaz et al., 2009).

      EMD Millipore’s QCM™ Gelatin Invadopodia Assays provide optimized materials and protocols to enable reproducible analysis of invadopodia in invasive tumor cells. All of the components necessary for affixing a thin film of fluorescent matrix to glass culture surfaces are provided. In addition, compatible reagents are provided for co-localizing the actin cytoskeleton and nuclei with invadopodial degradation sites. This assay may be used for assessing activity of inhibitors and promoters of invadopodia formation and function. Finally, different cell types as well as individual cells in heterogeneous populations may be analyzed for invasive potential with the QCM™ Gelatin Invadopodia Assay.
      Materials Required but Not Delivered1. Sterile cell culture hood

      2. Pipettors, liquid aspirators, etc. for handling of cells and liquid reagents

      3. Sterile plasticware (cell culture flasks, centrifuge tubes, pipettes, pipette tips, etc. for handling of cells and liquid reagents)

      4. Sterile glass substrate (e.g., chamber slide, coverslip, glass-bottom dish/multi-well plate)

      5. Sterile deionized water

      6. Sterile Dulbecco’s phosphate-buffered saline (DPBS), without calcium or magnesium

      7. 70% ethanol in sterile water

      8. Cell type of interest, with appropriate growth medium and cell detachment buffer (e.g., 0.25% trypsin)

      9. Hemocytometer (e.g. Scepter™ Handheld Automated Cell Counter)

      10. Trypan blue or equivalent viability stain

      11. Low speed centrifuge for cell harvesting

      12. CO2 tissue culture incubator

      13. 3.7% formaldehyde in DPBS (or equivalent) for cell fixation

      14. Methanol for phalloidin reconstitution

      15. Blocking/permeabilization buffer for phalloidin/DAPI staining (e.g., 2% blocking serum/0.25% Triton X-100 in DPBS)

      16. Slide mounting media (with anti-fade reagent) and cover glasses, if appropriate

      17. Microscope/image acquisition system (for phase contrast and fluorescence)

      18. Fluorescence filters for Cy3, FITC and DAPI imaging (see Table 2 for specific excitation/emission wavelengths)

      19. Image analysis software (e.g., NIH ImageJ)
      References
      Product Information
      Components
      • Cy3-Gelatin, 4X: One vial containing 0.5 mL
      • Unlabeled Gelatin, 4X: One vial containing 2 mL
      • DAPI: One vial containing 100 µL at 100 µg/mL in water
      • Poly-L-Lysine, 2X: One vial containing 5 mL
      • Glutaraldehyde, 16X: One vial containing 1 mL
      • FITC-Phalloidin: One vial containing 20 µg
      Detection methodFluorescent
      Applications
      ApplicationThis QCM Gelatin Invadopodia Assay (Red) provides the reagents necessary for affixing a thin, uniform layer of Cy3-labeled gelatin to a glass culture substrate, allowing for rapid detection of matrix degradation.
      Key Applications
      • Invasion Assay
      • Migration
      Application NotesThe EMD Millipore QCM™ Gelatin Invadopodia Assay (Red) provides the reagents necessary for affixing a thin, uniform layer of Cy3-labeled gelatin to a glass culture substrate, allowing for rapid detection of matrix degradation (Artym et al., 2009, Xu et al., 2009). A poly-L-lysine coating is first adsorbed to the glass substratum. The substrate is then treated with a dilute glutaraldehyde solution to bi-functionally “activate” the surface for further protein binding. Subsequent incubation of the surface with fluorescent gelatin allows covalent coupling between the poly-L-lysine and gelatin via reactive aldehyde (-CHO) groups. The fluorescently-coated glass is now prepared for cell culture by disinfection with 70% ethanol, followed by quenching of free aldehydes with amino acid-containing growth medium. Upon completion of fluorescent substrate preparation, cell types of interest may be seeded onto the gelatin surface for a desired amount of time. Depending on cell type, degradation may occur within a few to several hours. Treatment compounds of interest may also be introduced within the culture period.

      With EMD Millipore’s QCM™ Gelatin Invadopodia Assay (Red), degraded areas of gelatin, now devoid of fluorescence, may be microscopically visualized and quantified using image analysis software algorithms. The assay also provides fluorescent FITC-phalloidin and DAPI, for visualization of cytoskeletal F-actin and nuclei, respectively, to allow co-localization of degradation with cellular features. Potential activators or inhibitors of invadopodia formation may be investigated for their influence on the degree and frequency of matrix degradation, and the assay may be further combined with immunocytochemical staining for other molecules of interest in pathway studies.
      Biological Information
      Physicochemical Information
      Dimensions
      Materials Information
      Toxicological Information
      Safety Information according to GHS
      Safety Information
      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 ConditionsStore Cy3-Gelatin, Unlabeled Gelatin, Poly-L-Lysine and DAPI at 2-8°C. Store Glutaraldehyde and FITC-Phalloidin at -20°C. Use all reagents within 4 months from date of receipt.
      Packaging Information
      Material Size32 assays (1 kit)
      Material PackageEnough Reagents for 4 x 8-well Chamber Slides (32 assays)
      Transport Information
      Supplemental Information
      Specifications

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      Categories

      Life Science Research > Cell Analysis > Cell-based Assays > Cell Invasion Assays