4.12. Fluorescent Gelatin Degradation Assay

Oregon Green 488-labeled gelatin was from Molecular Probes (Invitrogen). Sterilized coverslips (18-mm diameter) were coated with 50 μg/mL poly-L-lysine for 20 min at RT, washed with PBS (phosphate buffered saline), and fixed with 0.5% glutaraldehyde for 10 min. After 3 washes with PBS, the coverslips were inverted on a 40-μL drop of 0.2% fluorescently labeled gelatin in 2% sucrose in PBS and incubated for 10 min at RT. After washing with PBS, coverslips were incubated in a 5 mg/mL solution of borohydride for 3 min, washed three times in PBS, and incubated with 1 mL of complete medium for 30 min. 5 × 10⁴ cells per 12-well were plated on the fluorescent gelatin-coated coverslips and incubated at 37 °C for 48 h. Cells were then washed three times with PBS and fixed with 4% PFA (paraformaldehyde) for 20 min and processed for labeling with Texas Red-Phalloidin and DAPI (diamino-2-phenylindole). The coverslips were mounted with Mowiol mounting medium. Cells were imaged on a confocal microscope (Leica TCS-SP5). The graph represents the percentage of degradative cells (n = 100 per experiment) compared to t = 0 of 3 independent experiments ± SD.