Quantikine human coagulation factor 3 tissue factor immunoassay

Plasma uPA and plasminogen activator inhibitor-1 (PAI-1) were analyzed using enzyme-linked immunosorbent assays (ELISA) as described previously (35 (link)). Briefly, plasma tissue factor (TF) was analyzed by ELISA using the Quantikine Human Coagulation Factor III/Tissue Factor Immunoassay (R&D Systems, Minneapolis, MN, USA). D-dimer in plasma samples was determined using the IMUCLONE D-Dimer ELISA (American Diagnostica, Greenwich, CT, USA) system. Plasma uPA was measured using the Human u-Plasminogen Activator/Urokinase Quantikine ELISA kit (R&D Systems). Plasma PAI-1 was measured using the Human Serpin E1/PAI-1 Quantikine ELISA Kit (R&D Systems).
Plasma samples were analyzed for 51 plasma cytokines and angiogenic factors (TGF-β1, TGF-β2, TGF-β3, IFN-α2, IL-1α, IL-2Rα, IL-3, IL-12p40, IL-16, IL-18, CTACK, Gro-α, HGF, LIF, MCP-3, M-CSF, MIF, MIG, β-NGF, SCF, SCGF-β, SDF-1α, TNF-β, TRAIL, IL-1β, Il-1RA, IL-2, IL-4, IL-5,IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-17, eotaxin, FGF basic, G-CSF, GM-CSF, IFN-γ, IP-10, MCP-1, MIP-1α, MIP-1β, PDGF bb, RANTES, TNF-α, and VEGF) using pre-designed panels as described previously and were available for subset of patients (Bio-Plex Pro TGF-β assay, Bio-Plex Pro Human Cytokine 21- and 27-plex immunoassays; Bio-Rad Laboratories, Hercules, CA, USA) (35 (link)).

HUVEC were seeded at a density of 400,000 cells/well in 6-well plates 24 h before stimulation. PBMC were seeded at a density of 11 to 12 million cells/well in 6-well plates followed by a 1-h adhesion period and removal of supernatants. HUVEC or adherent monocytes were incubated with the respective test compounds or vehicles for the indicated time periods. Thereafter, supernatants were removed and cells were lysed in Cell Lysis Buffer 1 (R&D Systems) for 45 min under shaking conditions (1400 rpm, 25°C). Afterwards, lysates were centrifuged at 14,000 g for 5 min at 4°C. Supernatants were used for measurement of TF protein that was quantified using the Quantikine^® Human Coagulation Factor III/Tissue Factor Immunoassay (R&D Systems) according to the manufacturer's instructions. TF protein was normalized to total protein amounts determined in the respective supernatant using the bicinchoninic acid assay (Pierce, Rockford, IL, USA).