Human vegfr1 flt 1 duoset elisa

The concentration of circulating antiangiogenic factors sFlt-1 and sENG, and the potent vasoconstrictor ET-1, were measured in maternal serum collected from patients diagnosed with early-onset preterm preeclampsia (n = 10), term preeclampsia (n = 4), and normotensive gestation-matched control patients (n = 17). Serum levels were measured using the Human VEGFR1/Flt-1 DuoSet ELISA (1/20 dilution; DY321B, R&D systems, Minneapolis, MN, USA), the Human Endoglin/CD105 DuoSet ELISA (1/15 dilution; DY1097, R&D systems), and the Endothelin-1 Quantikine ELISA Kit (neat serum; DET100, R&D systems, sensitivity: 0.207 pg/mL), according to the manufacturer’s instructions (intra-assay coefficients of variations under 10%).

Freshly obtained placental tissue slices were cut from three different areas of each placenta and washed three times in cold phosphate-buffered saline (PBS; Lonza, Walkersville, MD, USA), after which the decidua and chorionic plate were removed. Tissue containing chorionic villi was then cut in explant blocks of 2 × 2 mm. Explants from the three different areas were combined in one well in 2 mL DMEM/F12 medium (Gibco, Thermo Fisher Scientific, Paisley, UK) containing 10% FCS (GE Healthcare, Eindhoven, The Netherlands), 1.95 g/L NaHCO₃ and 100 μg/mL Primocin (Invivogen, San Diego, CA, USA) in 12-well plates, and equilibrated at 37 °C for 3 h at 8% O₂ and 5% CO₂. Thereafter, explants were transferred to a new plate and incubated with or without 100 μmol/L esomeprazole (Sigma-Aldrich) in the above medium for 24 h. sFlt-1 was measured in the medium using the human VEGFR1/Flt-1 DuoSet ELISA (R&D Systems, Minneapolis, MN, USA).