BOECs were isolated from healthy donors as described by Ramirez et al.24 (link). For all experiments, except the multi-omics and secretome experiments, three different pools of three unique BOEC donors (mixed sexes and ages) were used (Supplementary Table 2 ). For the multi-omics and secretome experiments, BOECs from three different donors (mixed sexes and ages) were pooled. Culture flasks and dishes were coated with collagen type I (50 µg/ml, BD biosciences) for 1 h prior to use. Cells were cultured in EC basal medium (Lonza) supplemented with 18% FCS (Bodinco) and EGM bulletkit (Lonza) unless stated otherwise. For SILAC labeling, BOECs were maintained for 5 passages as described by Beguin et al.36 (link) in custom-made EGM medium (Lonza), containing EBM2 medium (not containing Arganine and Lysine) (Lonza), EGM bulletkit (Lonza) and 18% FCS for passages 1–3 and in 18% 1 kDa dialyzed FCS for passages 4–5. Cells were SILAC labeled by the addition of isotope-labeled amino acids during all passages (light: Arg0 and Lys0, medium: Arg6 and Lys4, heavy: Arg10 and Lys8, Cambridge Isotopes). After five passage incorporation of labeled amino acids reached >95% in the total proteome.
Ec basal medium
Manufactured by Lonza
Sourced in United States
EC basal medium is a cell culture media formulation designed to support the growth and maintenance of endothelial cells. It provides the necessary nutrients and components for the in vitro culture of endothelial cell types.
Automatically generated - may contain errors
Lab products found in correlation
Mouse anti human cd31 microbeads, by Miltenyi Biotec (2 mentions)
Type 1 collagen, by BD (1 mentions)
Fetal calf serum (fcs), by Lonza (1 mentions)
Egm bulletkit, by Lonza (1 mentions)
Vascular endothelial growth factor (vegf), by Thermo Fisher Scientific (1 mentions)
Thp 1 cell, by American Type Culture Collection (1 mentions)
6 well plate, by BD (1 mentions)
Tnf α, by R&D Systems (1 mentions)
4 protocols using ec basal medium
1
Isolation and SILAC Labeling of BOECs
2
Endothelial Cell Chemotaxis Assay
3
Isolation and Characterization of HMVECs
4
Synovial Fibroblast Isolation and Characterization
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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.
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.
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