Mouse anti human cd31 microbeads

Fresh STs were minced and digested in tissue enzyme digestion solution as described previously [24 (link)]. The synovial fibroblasts were maintained in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% FBS. Cells were seeded in 6-well plates (BD Biosciences, Bedford, MA, USA) at a density of 1 × 10⁵ cells per well, and were maintained in complete medium. After overnight serum starvation, cells were treated with 25 ng/ml TNF-α (R&D Systems, Minneapolis, MN, USA) for 24 hours. Cell-conditioned medium and cell lysates were collected. We used fibroblasts from three NL knees; three knees and one hip from OA patients; and six knees from RA patients.
Human dermal microvascular endothelial cells (HMVECs) were purified from digested skin biopsies using mouse anti-human CD31 MicroBeads (Miltenyi Biotec, Cambridge, MA, USA), according to the manufacturer’s protocol. Cells were cultured in EC basal medium (Lonza, Walkersville, MD, USA) with growth factors. In order to confirm EC purity, we used antibodies to EC markers von Willebrand factor and CD31 and immunohistochemistry. THP-1 cells (human acute monocytic leukemia cell line) were purchased from the American Type Culture Collection (Manassas, VA, USA). THP-1 cells were cultured in RPMI supplemented with 10% FBS.