Fluorescent gelatin matrix degradation assays

For the preparation of fluorescent-gelatin-coated coverslips, a gelatin/sucrose solution (2.5% each) was freshly prepared in PBS and kept at 37°C until usage. Glass coverslips (12 mm diameter) were washed with 20% nitric acid for 30 min at room temperature (RT). After washing thoroughly with deionized water, the coverslips were pre-coated with 50 μg/ml poly-L-lysine (#BSBTAR0003, BOSTER Bio) in dH₂O for 20 min at RT, washed once with PBS, and incubated with ice-cold 0.5% glutaraldehyde in PBS on ice for 15 min. Coverslips were then washed 3x with ice-cold PBS and incubated with 125 μg/ml Oregon-Green-488-conjugated gelatin (#{"type":"entrez-nucleotide","attrs":{"text":"G13186","term_id":"1125046","term_text":"G13186"}}G13186, Thermo Fisher Scientific) diluted in gelatin/sucrose solution for 15 min at RT in the dark with gentle rocking. Excess gelatin was washed off with PBS and glutaraldehyde was quenched with 5 mg/ml NaBH₄ in dH₂O for 15 min at RT. After three washes with PBS, the coated coverslips were sterilized with 70% EtOH for 30 min at RT and stored in sterile PBS in the dark until use.

For the matrix degradation experiments, 5-10 × 10⁴ cells were seeded on the coated coverslips for 24 h, fixed with 4% formaldehyde in PBS for 10 min at RT and counterstained with 0.1 μg/ml DAPI and 15 nM Phalloidin Fluor 647 for 1 h at RT, and mounted using Dako fluorescent mounting medium (#S3023, Agilent/Dako). Relative gelatin degradation was quantified using ImageJ. Data shown are representative of at least 3 replicate experiments. Data are presented as mean ± SD. ^∗ p < 0.05, ^∗∗ p < 0.01, ^∗∗∗ p < 0.005.