Human igf 1 duoset

To characterize the paracrine activity of ASCs, conditioned medium was collected, centrifuged at 2,500 g to remove cell debris, and stored at –70 °C until the measurements were taken. To detect secreted proteins, conditioned medium was analyzed using the Proteome Profiler Human Angiogenesis Array Kit (R&D, USA) and Proteome Profiler Human Protease Array Kit (R&D, USA), according to the manufacturer’s instructions. The data were analyzed using Image Lab™ Software Version 5.0 (Bio-Rad, USA). VEGF-α, TGF-β, IL-6, IL-8, MCP-1, and IGF-1 concentrations in ASC conditioned medium were evaluated using the Human VEGF ELISA Set (Peprotech, USA), Human TGF-β1 DuoSet ELISA (R&D, USA), Human IL-6 ELISA Set (BD, USA), Human IL-8 ELISA Set (BD, USA), Human CCL2/MCP-1 DuoSet (R&D, USA), and Human IGF-1 DuoSet (R&D, USA), according to the manufacturer’s instructions.

Using a 21-gauge needle and Becton Dickinson Vacutainer (SST) tubes, 5 mL of blood were collected from the cardinal veins in the forehand of each participant before and after the 16-week intervention. The blood samples were centrifuged at 3000 rpm for 15 min and then stored at −80 °C until they were analyzed. The analysis of the serum levels of BDNF, VEGF, and IGF-1 was performed using a sandwich enzyme-linked immunosorbent assay (ELISA). For BDNF, we used the commercially available human BDNF Kit (#DBD00; R&D Systems, Minneapolis, MN, USA, datasheet available at https://resources.rndsystems.com/pdfs/datasheets/dbd00.pdf); for VEGF, the human VEGF Kit (#DVE00; R&D Systems, Minneapolis, MN, USA, datasheet available at https://resources.rndsystems.com/pdfs/datasheets/dve00.pdf); and for IGF-1, the human IGF-1 DuoSet (#DY291; R&D Systems, Minneapolis, MN, USA, datasheet available at https://resources.rndsystems.com/pdfs/datasheets/dy291.pdf). A micro-plate reader (Emax; Molecular Devices, San Jose, CA, USA) was used to measure absorbance at 450 nm for quantification.

Eight milliliters of blood were collected from the antecubital vein of each subject before and after 16 weeks of intervention, using a 22-gauge needle and a serum separator tube (Becton Dickinson, Franklin Lakes, NJ, USA). Collected blood samples were centrifuged for 15 min at 3000 rpm and then stored at −80 °C until analysis. The analysis of serum BDNF, VEGF, and IGF-1 levels was performed using sandwich enzyme-linked immunosorbent assay (ELISA). For BDNF, we used the human BDNF Quantikine Kit (Catalog No. DBD00; R&D Systems, Minneapolis, MN, USA); for VEGF, we used the human VEGF Quantikine Kit (Catalog No. DVE00; R&D Systems), and for IGF-1, we used the human IGF-1 DuoSet (Catalog No. DY291; R&D Systems). Fluorescence was measured at 450 nm with a microplate reader (Emax; Molecular Devices, Sunnyvale, CA, USA).