IM MNC-conditioned media contain elevated gelatinolytic activity, which is inhibited by an MMP inhibitor. (A) The gelatinolytic activities of media conditioned by PB MNCs from healthy volunteers (n = 6), patients with PV (n = 8), and patients with IM (n = 11); purified human NE; MMP-9; or PBS were analyzed by zymography. MNCs were incubated in serum-free IMDM at 37°C and 5% CO₂ for 48 hours, and the cell-conditioned media or purified proteases were electrophoresed in 10% acrylamide containing 2 mg/mL gelatin in a cold room. Following electrophoreses, the gels were washed with 2.5% Triton X-100, renatured for 36 to 48 hours at 37°C, and stained with Coomassie blue. (B) Effect of the MMP inhibitor, o-phenanthroline, on the gelatinase activity of the media conditioned by the MNCs isolated from healthy volunteers (n = 6), patients with PV (n = 8), and patients with IM (n = 11); purified human NE; MMP-9. The parallel gels were incubated in the presence of 1.0 mM o-phenanthroline after electrophoresis. (C) Densitometric analysis of gelatinolytic activities (MMP-9). The intensities of the bands in the conditioned media by IM and PV were quantitated relative to the normal control and expressed as fold increase.