Human tissue digestion and mast cell isolation

Fat was removed from the synovium and the synovium was cut into small pieces using sterile scissors. The synovium was then incubated in RPMI 1640 medium (Thermo Scientific) containing 1 mg/mL collagenase I (Sigma), and 0.1 mg/mL DNase I (Roche). Enzymatic digestion was carried out for 1 h at 37 °C, after which the suspension was filtered through gauze and centrifuged at 450×g for 10 min. Pellets were washed with MACS buffer (PBS containing 5% FBS and 2 mM EDTA), and then filtered through a 100 μm and 70 μm cell strainer (Jet Biofil) to obtain a single-cell suspension. CD117-positive cells were isolated from synovial single-cell suspensions using a human CD117 microbead kit according to manufacturer’s instructions (Miltenyi Biotec, 130-091-332). The sorted cells were used only when their viability was determined by trypan blue exclusion staining and their purity determined by tryptase staining was > 90%.