QCM Gelatin Invadopodia Assay (Red)

The 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.

Enough Reagents for 4 x 8-well Chamber Slides (32 assays)

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

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.

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