Human vegf duoset elisa

For VEGF ELISA, PBMC and CBMC were stimulated for 16–22 h with anoxia or E. coli. Afterwards, supernatants were collected and stored at −80 °C until further analysis. ELISA was performed using the Human VEGF DuoSet ELISA and DuoSet Ancillary Reagent Kit 2 (both from R&D Systems, Minneapolis, United States of America) following the manufacturer’s instructions. For each sample duplicates were measured and the mean of the optical density was converted into the amount of VEGF in pg/ml based on the values derived from the standard curve.

Human VEGF DUOSET ELISA (R&D System) was used to measure VEGF-A levels in culture medium according to the manufacturer’s instructions. Absorbance was measured at 450 nm using a GloMax Explorer plate reader (Promega). Graphs show an average of three experiments.

Fibrin gels (70 μL; n = 4 gels per group) were made of 10 mg/mL fibrinogen, 2 U/mL thrombin, 4 U/mL Factor XIIIa, and 5 mM CaCl₂ in HEPES buffer, in which 1 μM of α2PI and 200 ng VEGF-A-PlGF-2_123–144 and PDGF-BB-PlGF-2_123–144 were added, were incubated for 1 h at 37 °C with 5% CO₂. Then, the fibrin gels were transferred into a 24-well cell culture plates and incubated in 1 ml release buffer (Tris 20 mM, NaCl 150 mM, 0.1% BSA, Pen/Strep, pH 7.4) containing 2.5 nM plasmin (Roche, #10602361001). The plate was kept at 37 °C with 5% CO2 until the gels were fully degraded. The plasmin-containing buffer was daily replaced, collected and stored at −20 °C. The daily release of VEGF-A was quantified by ELISA (Human VEGF DuoSet ELISA, R&D systems) as instructed by the manufacturer and normalized to the total released amount.

ARPE-19 and RPE supernatants were collected after treatment with LH extracts (1, 10, 50, and 100 µg/mL) on Day 3 after stimulation [11 (link)]. Medium with sulfated fucan was exchanged 24 h (ARPE-19) or 4 h (RPE) before collecting supernatants. Human VEGF DuoSet^® ELISA was used for ARPE-19, whereas the Human VEGF Quantikine^® ELISA was used for RPE supernatants (both R&D Systems, Wiesbaden, Germany). ELISAs were performed according to the manufacturer’s instructions. Untreated cells and medium samples were tested as controls. To set the VEGF secretion in relation to the cell viability, MTT assay was conducted after collection of the supernatants on Day 3.

To determine VEGF levels in cell culture-conditioned medium, medium was collected from culture wells and frozen 72 h after treatment start. VEGF secretion was determined using quantitative-sandwich ELISA kit (Human VEGF DuoSet ELISA, R&D Systems, #DY293B) following manufacturer’s instructions. Normalization of VEGF secretion was performed proportionate to the number of cells by dividing the result of VEGF secretion by % viability (from control) according to a PrestoBlue viability assay.

VEGF-A levels in CM of ascTAM or asc-MDM stimulated with PGI₂ analog or solvent control (DMSO) were quantified by ELISA (Human VEGF DuoSet ELISA, R&D Systems) according to the manufacturer’s instructions.

Presence of vascular endothelial growth factor (VEGF) on ASC bioproducts was determined by solid phase sandwich ELISA using the human VEGF DuoSet ELISA (R&D Systems, USA) according to manufacturer’s instructions. The samples were read immediately at 450 nm with a wavelength correction at 570 nm using a VICTOR X4 multilabel plate reader (Perkin Elmer, Waltham, Massachusetts, USA). Levels of cytokines were quantified against an eight-point standard curve using twofold serial dilutions in reagent diluent.