Nunc lab tektm chambered coverglasses

To visualize invadopodia matrix degradation, 5x10⁴ cells were embedded in 200 μl of Matrigel containing 25 μg/ml DQ-type I collagen (Life Technologies, Invitrogen) and mixed gently before plating in 8 wells Nunc^TM Lab-Tek^TM chambered coverglasses (Thermo Scientific) and incubating at 37°C for 2 hours. Then, medium supplemented with 10% FetalClone III and 2 ng/ml TGF-β was added on top of the Matrigel. After 72 hours, cells were fixed with 4% paraformaldehyde (PFA), permeabilized with 0.5% saponin, blocked with 5% bovine serum albumin (BSA), and F-actin was stained with TRICT-conjugated phalloidin (Sigma-Aldrich). Invadopodia were identified as actin-rich protrusions that colocalized with matrix degradations (enhanced DQ-type I collagen signal). 3D reconstructions were generated from Z-stacks using Volocity Software (Perkin Elmer, Waltham, MA).

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.