Cell proliferation elisa

LEC were seeded in 96-well plates in EGM 2-MV medium at a density of 4×10³cells/well. On day 2, medium was replaced with serum-free medium for 4 hours. Wells were washed with PBS and 100 µl/well of EMB-2 control medium or MSC conditioned medium was added in the presence of BrdU (10 µl/ml; Cell Proliferation ELISA, BrdU, Roche, Mannheim, Germany) and 1% FBS. Cells were fixed and stained after 48 h according to manufacturer's instructions (Cell Proliferation ELISA, BrdU, Roche, Mannheim, Germany). For WST-1 assay (Roche, Mannheim, Germany), the same protocol was used. Control medium and MSC conditioned medium were pre-incubated for 1 h at 37°C with 1 µg/ml soluble VEGF Receptor-1, -2 (sVEGFR-1 or -2) (R&D Systems, Abingdon, UK) or ZM 323881 (Tocris Bioscience, Bristol, UK) at a concentration of 10 nM. After a 2 h incubation with WST-1 reagent, sample absorbance was measured according to manufacturer's instructions. Colorimetric analysis was performed with an ELISA reader (Multiskan FC, Thermoscientific, Waltham, MA).

Proliferation of primary cultured pulmonary artery and thoracic aorta SMCs was assessed using a colorimetric Cell Proliferation ELISA (Roche, Mannheim, Germany), evaluating 5-bromo-2’-deoxyuridine (BrdU) incorporation during DNA synthesis in replicating cells. Briefly, pulmonary artery and thoracic aorta SMCs in DMEM supplemented with 10% FCS were seeded in 96-well plates at a density of 104 cells/well and allowed to adhere for 24 h. Cells were subjected to 24 h of growth arrest in FCS-free DMEM and then treated with increasing concentrations of recombinant mouse chemerin (5.10^–9, 10^–8, 5.10^–8, and 10^–7 mol/L) with or without endothelin-1 (10^–7 mol/L) in FCS-free DMEM and diluted BrdU labeling reagent (1:100). After a 24-h incubation period, BrdU incorporation was revealed using an anti-BrdU-peroxidase antibody, according to manufacturer’s recommendations (Cell Proliferation ELISA, Roche) and absorbance was measured using a microplate reader (Packard, Canberra, Australia) at 370 nm. Proliferation levels were expressed as relative fold increase over the mean value of the proliferative rate of the basal condition (corresponding to FCS-free DMEM) fixed to 0.

BMM were cultured for 24 h in a 96-well plate (5000/well). Cells were synchronized by 24 h serum deprivation, afterwards cell growth was initiated over night by adding serum containing growth medium. Proliferation was quantified by determining BrdU incorporation after 24 h (Cell Proliferation ELISA, BrdU (colorimetric), Roche, Diagnostics GmbH, Mannheim, Germany).
Osteoblast-like cells were seeded in a 96-well plate (5000/well) and cultured in osteogenic medium for either 13 or 20 days. Subsequently, proliferation was determined via BrdU incorporation for 24 h (Cell Proliferation ELISA, Roche).