Fluorescently labeled gelatin has been an informative tool in the study of cell invasion and matrix degradation. The method involves plating cells on a culture surface coated with a thin layer of a fluorescently labeled matrix and then visualizing regions where the cell has degraded the matrix to cause a loss of fluorescence signal. Such assays have pinpointed cellular regions that initiate invasion and revealed that invasive cells extend small protrusions of localized protease activity, termed podosomes in nonmalignant cells and invadopodia in cancerous cells.
This invasion of cancerous cells through extracellular matrix layers is a key step in tumor metastasis, inflammation, and development. The process 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. Each of these invasion stages is executed by a suite of proteins, including proteases, integrins, GTPases, kinases, and cytoskeleton-interacting proteins.
The classical method for analyzing this process involves the application of cells to one side of a layer of gelled matrix molecule and quantifying the relative number of cells that traverse across the layer. Though such methods are useful for analyzing invasion at the cell population-level, fluorescently labeled gelatin has allowed for more detailed analysis of subcellular events.
However, conjugating fluorescent molecules to gelatin is laborious and dependent on user technique to create a homogenously labeled matrix. Inconsistent application on glass substrates is also possible due to non-standardized protocols.
QCM™ Gelatin Invadopodia Assay kits from EMD Millipore address these issues, providing a simplified and standardized method for producing homo-genously fluorescent matrices. The kits provide the reagents necessary for affixing thin, consistent coatings of pre-labeled fluorescent gelatin (fluorescein- or Cy3-conjugated) on glass substrates. They also include fluorescently labeled phalloidin (TRITC- or FITC-conjugated) and DAPI for visualizing cytoskeletal F-actin and nuclei, respectively, to allow for co-localization of matrix degradation with cellular features.
To demonstrate the utility of these kits in the visualization and quantification of gelatin degradation, multiple cell types were tested at multiple time points and following treatment with modulators of invadopodia formation.