Sensolyte 520 mmp14 assay kit

To assess MMP14 activity the SensoLyte 520 MMP14 Assay Kit (AnaSpec) was used. CAFs were obtained as previously described, then they were washed with PBS and harvested in assay buffer containing 0.1% Triton-X 100 and incubated for 10 min on ice. Further, samples were centrifuged at 2500×g for 10 min at 4 °C, and supernatants were transferred into fresh tubes. Protein concentration was measured using a BCA assay. Samples with the same amount of protein (ca. 10 µg) were incubated with an activator for 2 h at 37 °C. Next, the substrate was added, and the enzymatic reaction was performed for 30 min at 37 °C, and then stopped with a stop solution. The fluorescence of the product was measured (Ex/Em = 475/500 nm) using the GloMax Discover plate reader (Promega). For each experiment, the MMP14 activity was calculated compared to the control cells. Control was set as 100% of activity. Experiments were conducted in three repetitions; each condition was performed in two replicates.

To evaluate MMP14 activity, the SensoLyte 520 MMP14 Assay Kit (AnaSpec, Fremont, CA, USA) was used according to the manufacturer’s protocol. Briefly, cells were seeded in 60 mm Petri dishes and washed with PBS after 24 h, and then collected in assay buffer. Lysates were centrifuged at 4 °C for 10 min at 2500× g, then supernatants were transferred to fresh tubes. The protein content was determined with the standard Bradford method [55 (link)], followed by preparation of samples, each containing 30 µg of protein. To activate the MMP14, samples were incubated for 2 h at 37 °C in the presence of 1 nM of APMA (4-aminophenylmercuric acetate). Then, MMP14 substrate was added to start an enzymatic reaction, and, after 30 min of incubation at 37 °C, the reaction was stopped with a stop solution. Recombinant MMP14 (Merck Millipore, Burlington, MA, USA) was used as a positive control. The fluorescence of digested substrate was measured at 490/520 nm using Infinite M1000 Pro (Tecan, Männedorf, Switzerland) and i-control software (Tecan, Männedorf, Switzerland). The results were background corrected and calculated as a fold change of activity between the parental and resistant lines.

To assess MMP14 activity the SensoLyte 520 MMP14 Assay Kit (AnaSpec) was used. Cells were seeded into 6-well plates and activated towards CAKs as previously described. After seven days of keratinocytes incubation with melanoma CM or with melanoma on Transwell inserts, cells were rinsed with PBS, harvested in assay buffer containing 0.1% Triton-X 100 and incubated for 10 min at 4 °C. Samples were then centrifuged for 10 min at 4 °C at 2500×g, and supernatants were transferred into fresh tubes. Protein concentration was evaluated using a standard BCA assay. Samples with the same amount of protein (30 µg) were incubated with an activator for 2.5 h at 37 °C. Next, the substrate was added and the enzymatic reaction was conducted for 30 min at 37 °C and stopped with a stop solution. The fluorescence of the product was measured (Ex/Em = 475/500 nm) using a GloMax Discover plate reader (Promega). Control was set as 100% of MMP14 activity. Experiments were performed three times, each condition in two replicates.

MT1-MMP activity was performed as described previously [10] (link), [21] . Briefly, cells were lysed with a hypotonic buffer, then sonicated and centrifuged at a high speed (15,000 g) for 30 min. The cell lysates were assessed by a commercially available fluorescent assay kit (SensoLyte 520 MMP-14 assay kit, AnaSpec, San Jose, CA, USA) according to the manufacturer's instructions.