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.
Preparation and Cell Seeding
The QCM Gelatin Invadopodia Assay kits can accommodate 8-well glass chamber slides, glass multiwell plates, and glass coverslips. In this study, 8-well glass chamber slides were used, coated with dilute poly-L-lysine in deionized water and rinsed with Dulbecco’s PBS (DPBS). Next, dilute glutaraldehyde in DPBS was added to each well to “activate” the poly-L-lysine surface for further protein attachment. Each well was again rinsed with DPBS. Finally, 200 µL of dilute gelatin in DPBS, mixed at a 1:5 ratio of fluorescently labeled:unlabeled gelatin, was coated onto each well, followed by additional DPBS rinses.
To prepare for cell plating, the gelatin substrates were disinfected with 70% ethanol. After ethanol removal and rinsing in DPBS, free aldehydes were quenched by the addition of amino-acid-containing growth media. Cell types of interest were detached using 0.25% trypsin-EDTA, pelleted, then resuspended in growth medium to a concentration of 28,000 cells/mL (20,000 cells/cm2). Cells were seeded in a volume of 500 µL/well and cultured for the desired duration of degradation, generally between 8–48 hours.
Fixation, Staining, and Imaging
At the desired time-point, growth media was removed from the chamber slides, and samples were fixed with 3.7% formaldehyde in DPBS. Samples were then rinsed with fluorescent staining buffer.
For immuno-co-localization studies, primary antibody in fluorescent staining buffer was added to each well for incubation. Samples were then rinsed with fluorescent staining buffer before proceeding on to incubation with fluorescent secondary antibody, fluorescently conjugated phalloidin, and DAPI in staining buffer. Primary and secondary antibodies were omitted for stains incorporating phalloidin and DAPI only. Samples were then rinsed with fluorescent staining buffer and DPBS.
Mounted cover glasses were allowed to hard-set before fluorescent imaging with illumination and filters appropriate for fluorescein/FITC, Cy3/TRITC, and DAPI excitation and emission wavelengths. Samples were imaged on an inverted wide-field fluorescent microscope at 20X objective magnification for quantification studies or at 63X objective magnification for co-localization experiments. Image analysis was performed utilizing free, downloadable ImageJ software distributed by the NIH.