Human vegf duoset

Cytokine and growth factor releases were assessed by ELISA. The quantification of IL-6, IL-8, IL-10, and VEGF proteins at 4, 7, and 10 days in conditioned supernatants was assessed using respectively human IL-6, IL-8, IL-10 and human VEGF Duoset^® (R&D systems, Lille, France). Absorbance was measured at 450 nm with correction of non-specific background at 570 nm according to the manufacturer’s instructions.

The amount of IL-1β, IL-6, VEGF and BDNF secreted by astrocytes after the treatment with MS and healthy CSF were determined to confirm our qPCR results. Conditioned media of the experiment mentioned above was used for this purpose. Human IL-1β/IL-1F2 DuoSet, Human VEGF DuoSet, Human Total BDNF Quantikine ELISA kits (R&D Systems, USA) and Human Interleukin-6 ELISA Kit (SUNLONGBIOTECH, China) were used for this purpose, following their manufacturer’s protocol. Optical density was measured at 450 nm with correction at 570 nm. Concentrations were calculated using standard curve equations for each set and then analyzed using GraphPad Prism statistical analysis software.

Four PRP hyaluronan collagen composite matrix constructs of each of the 4 volunteers were cultured in vitro over a period of 8 days in 1 mL autologous plasma.
Concentrations of the growth factors PDGF, TGFβ1, and VEGF were measured by ELISA technique at 0 h, 8 h, 12 h, 24 h, 48 h, and 192 h (8 days) using kits from R&D Systems: Human PDGF-AB DuoSet (DY222), Human TGFβ1 DuoSet (DY240), and Human VEGF DuoSet (DY293B). Results of cultured empty control scaffolds were subtracted from the growth factor concentrations obtained from the cultured PRP loaded scaffolds in order to exclude an influence of the remaining small growth factor activity in the autologous plasma.

HK-2 cells were placed in 24 well plates (75 × 10³ cells/well) for 24 h. Controls received only medium. After the different treatments, the medium was removed and kept at −80 °C for ELISA assays. VEGF was analyzed using human VEGF DuoSet (R&D Systems, Minneapolis, MN, USA). VEGF mouse capture antibody diluted in phosphate-buffered saline (PBS) (pH 7.4) was incubated onto each well of a 96-well plate overnight (RT). Plates were washed three times between each step with PBS-Tween (0.05%). After blocking with bovine serum albumin (1%) in PBS for 1 h (RT), recombinant human VEGF standards diluted in blocking solution, or protein sample was added to each well. After incubation, 100 μl of biotinylated goat anti-human VEGF was added to each well, and plates were left for 2 h. Streptavidin-HRP 1:200 diluted in PBS was added and plates were incubated at room temperature for 20 min. Then, a H₂O₂ and tetramethylbenzidine (1:1) solution was added, protected from light and incubated for 20 min at room temperature. The reaction was stopped with 2 NH₂SO₃ and absorbance was immediately read at 450 nm. Samples were analysed by triplicated^{62 (link)}.