Reagent diluent concentrate 2 dy995

HUVECs were seeded in 12-well plates with EBM-2 Basal medium (CC-3156, Lonza) supplemented with EGM-2 SingleQuots Supplements (CC-4176, Lonza) for 24 h. Cells were then stimulated with 5 ng/ml bFGF (Biolegend) or 5 ng/ml bFGF in combination with either 50 or 100 µm K5 for 60 min. The cells were rinsed with PBS and lysed in cell lysis buffer containing IC diluent #12 (Reagent diluent concentrate 2 DY995, R&D Systems, Minneapolis, MN, USA, in distilled water), plus 10 µg/ml aprotinin (Ref.4139, R&D Systems, Minneapolis, MN, USA) and 10 µg/ml leupeptin (Ref.1167, R&D Systems, Minneapolis, MN, USA). FGFR1 phosphorylation was measured by a sandwich ELISA (DYC5079-2, R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions.

Serum ANA were assessed by indirect immunofluorescence in HEp-2 cells (Euroimmun, Germany). Rheumatoid factor (RF) was measured in immunoassays with the Dimension Vista® System (Siemens Healthcare Diagnostics, Germany). Anti-neutrophil cytoplasmatic antibodies (ANCA) were assessed with indirect immunofluorescence in a composite substrate of ethanol and formalin-fixed granulocytes (EOH) and HEp-2 cells, (Euroimmun, Germany). Anti-double-stranded DNA (dsDNA) antibodies were measured with an anti-dsDNA-NcX-ELISA (Euroimmun, Germany).
Serum concentrations of human sICAM-1 (DY720), sELAM-1 (DY72), and sVCAM-1 (DY809) were analyzed by commercially available ELISA (Duo-Set-Kit (R&D Systems, Germany). Reagent diluent concentrate 2 (DY995) and the substrate reagent Pack (DY999) were also purchased from R&D Systems. Sulphuric acid solution (35276) and phosphate buffered saline (P4417) were purchased from Sigma Aldrich (Germany). High binding 96-well ELISA plates (655081) were obtained from Greiner (Germany). Soluble adhesion molecule parameters for age- and gender-matched HC (n = 75) were determined in sera from healthy blood donors, after obtaining written, informed consent.