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The synovial tissues were minced and treated for 4 h with 2.5 mg/mL of type I collagenase (Sigma Aldrich, Saint Louis, MO, USA) in Dulbecco's modified Eagle's medium (DMEM) (Euroclone, Italy) at 37°C in 5% CO2. Dissociated cells were then centrifuged at 1000 ×g, resuspended in DMEM supplemented with 10% fetal calf serum (FCS) (Fetalclone1 Hyclone Logan, UT, USA), 2 mM L-glutamine, 100 units/mL penicillin, and 100 mg/mL streptomycin (Euroclone, Italy), and plated in 75 cm2 flasks (Primo Cell Culture Flask, Euroclone, Italy). After overnight culture, nonadherent cells were removed, and adherent cells were cultivated in DMEM supplemented with 10% FCS. The cultures were kept at 37°C in 5% CO2, and the medium was replaced every 3 days.
The purity of the cells was tested by flow-cytometric analysis using phycoerythrin-conjugated anti-CD14 (PharMingen, San Diego, CA, USA) and fluorescein isothiocyanate phycoerythrin-conjugated anti-CD3, anti-CD19, anti-CD14, or anti-Thy-1 (CD90) monoclonal antibodies (R&D Systems Minneapolis, MN). A FACS Calibur flow cytometer (488Ex/620Em) (Becton Dickinson, San José, CA, USA) was used for the analysis. At passage 3, the cells were morphologically homogeneous and exhibited the appearance of FLS, with typical bipolar configuration under inverse microscopy. Most cells (>98%) expressed the surface markers for fibroblasts (Thy-1) and were negative for the expression of CD3, CD19, and CD14. Synoviocytes from passages 3–8 were used in each experiment. |
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