Key Specifications Table
|Key Applications||Detection Methods|
|Invasion Assay, Migration||Fluorescent|
|Description||QCM™ Gelatin Invadopodia Assay (Red)|
|Overview||Read our application note in Nature Methods!
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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 Delivered||1. 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)
|Safety Information according to GHS|
|Storage and Shipping Information|
|Storage Conditions||Store 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.|
|Material Size||32 assays (1 kit)|
|Material Package||Enough Reagents for 4 x 8-well Chamber Slides (32 assays)|