2.7. Detection of Metalloproteinase Activity by Zymography

The activity of gelatinases MMP-2 and MMP-9 was determined using gelatin zymography. Cell supernatants (15 μL), CSF (15 μL) or exosomes (20 μg) were mixed 1:4 with 4X NuPage LDS sample buffer (11836170001, Roche (Basel, Switzerland)) (without β-mercaptoethanol) and loaded onto 10% SDS-PAGE gels that had been supplemented with 3 mg/mL gelatin (Sigma Aldrich (St. Louis, MO, USA). Recombinant MMP-2 (Sigma Aldrich (St. Louis, MO, USA), 86607) was also added as a reference marker. Electrophoresis was used to separate proteins according to their molecular weight. Subsequently, to restore MMP activity, gels were incubated in zymogram renaturing buffer (Thermo Fisher Scientific (Waltham, MA, USA) at room temperature for 30 min. Renaturing buffer was removed and replaced with a zymogram developing buffer (Thermo Fisher Scientific (Waltham, MA, USA) and incubated overnight at 37 °C, during which time gelatinases degrade the gel. This degradation was visualised by the staining of the gels with 0.5% w/v Coomassie blue R-250 in 40% v/v methanol, 10% v/v glacial acetic acid (Bio-Rad (Hercules, CA, USA) at room temperature for 15 min. To reduce background staining, gels were washed in a destaining solution (40% v/v methanol, 10% v/v glacial acetic acid) for two hours to reveal the gelatinase bands. Gels were fixed in a fixative solution (2% paraformaldehyde, 0.075 M lysine, and 0.01 M sodium periodate, pH 7.4) for 15 min before being dried and photographed.