2.8. MMP2/9 Activity Assay

InnoZyme™ Gelatinase (MMP-2/MMP-9) Fluorogenic Activity Assay kit (Millipore Sigma, Burlington, MA, USA) was used to assess the activity of MMPs2 and MMP9 in cell culture media. This assay utilizes a triple-helical collagen-like peptide that is highly selective for MMP2 and MMP9. This approach relies on the fluorescence resonance energy transfer (FRET) phenomenon. The fluorescence of the FRET peptide is quenched while it is intact and released only when the peptide is cleaved. Accordingly, this peptide is used as an indicator of MMP2/9 activity. The manufacturer protocol was followed. Briefly, conditioned media from different treatments were passed through 0.2 μm depth filters to get rid of cells and debris and then adjusted to the same protein concentration in all samples. Pro-MMP2 and pro-MMP9 were pre-activated by incubation with p-Aminophenylmercuric Acetate (APMA) followed by incubation with the substrate at 37 °C for 6 h. Finally, fluorescence was measured via multimode plate reader (M3 Molecular Devices) with excitation and an emission wavelength of 320 and 405 nm, respectively.