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Purification of YKL-40. SVF Inauhzin were incubated with serum-free DMEM for 24 h in collagen gels. The CM of 300 ml was fractionated by a 10-ml Gelatin-Sepharose 4B (GE Healthcare, Uppsala, Sweden) column. The non-gelatin-bound fraction was then passed through a 5-ml Hitrap Heparin HP column (GE Healthcare) equilibrated with 0.1 M phosphate buffer (pH 7.4). The heparin-bound material was eluted sequentially with 0.1 M phosphate buffer (pH 7.4) containing 300, 400, 500 mM, and 1 M NaCl with an AKTAprime chromatography system (GE Healthcare). Fractions eluted by 400 mM NaCl were concentrated with Centriplus YM-3K (Millipore).
Measurement of collagenolytic activity. The assay was performed as directed by the protocol of the Type I Collagenase Activity Assay Kit (LIFE, Yamagata, Japan) with some modifications. Briefly, mixtures with final concentrations of 250 μg/ml of FITC-labeled type I collagen from calf skin, various concentrations of purified YKL-40, human MMP-1 active form, and ice cold 1× neutralization buffer supplied with the kit in 200 μl were incubated at 37 °C for 1 h. Samples were denatured with 200 μl of denature/stop solution supplied with the kit for 30 min at room temperature and then centrifuged at 6000g for 15 min. The fluorescence of the supernatant was measured with a primary meristems fluorescence spectrophotometer.