Alexa fluor 488 conjugated gelatin

Alexa-Fluor-488-conjugated gelatin and fibrinogen were from Life Technologies and FITC-conjugated hyaluronic acid (HA) from Sigma. Fibronectin (Life Technologies) and laminin (R&D Systems, Abingdon, UK) were conjugated to Alexa-Fluor-488 and Alexa-Fluor-555 fluorophores by using protein-labeling kits according to manufacturer's instructions (Life Technologies). Acid-washed coverslips were coated overnight with matrix components, then washed and blocked in RPMI with 10% FCS before plating with cells. The use of fluorescent labels allowed confirmation of the uniform distribution of the matrix components across the coverslip. Podosomes were labelled with Alexa-Fluor-555 or -633-phalloidin and anti-vinculin/Pacific Blue-rabbit anti-mouse IgG (Life Technologies).

Nunc^TM Lab-Tek^TM chambered coverglasses (Thermo Scientific) were coated with 50 μg/ml poly-L-lysine for 15 minutes, washed with PBS, and cross-linked with 0.5% glutaraldehyde for 15 minutes. Glutaraldehyde was removed and a 100-μl drop of 1 mg/ml Alexa Fluor 488-conjugated gelatin (Life Technologies, Invitrogen, Barcelona, Spain) was added for 10 minutes to each coverglass. The fluorescent signal was quenched with 5 mg/ml sodium borohydride for 3 minutes followed by a PBS wash. Finally, coverglasses were sterilized with 70% ethanol for 5 minutes and covered with cell growth medium 1 hour before use. To detect invadopodia formation, 100 cells/μl cells were seeded over cross-linked fluorescent conjugated matrix for 1 to 120 hours. The percentage of cells showing degradation was quantified by microscopy inspection. A cell was considered positive for the presence of invadopodia when it has at least one actin/cortactin enriched cell protrusion inside of an individual or sets of degraded areas in the ventral side of the cells.