Human ccl2 mcp 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.

Cell culture media was collected and soluble factors expression was analysed 24 h after initiation of cell culture. The levels of IL6, IL8/CXCL8, CCL2, IL1B, IL2, IL4, IL12, TNFα and IFNγ secreted into the growth medium were measured using Human IL-6 DuoSet ELISA Development Kit (R&D System, Wiesbaden, Germany), Human IL-8 Standard ABTS ELISA Development Kit (Peprotech, Rock Hill, NJ, USA), Human CCL2/MCP-1 DuoSet (R&D System), Human IL-1β/IL-1F2 DuoSet ELISA Development Kit (R&D System, Wiesbaden, Germany), Human IL-2 ELISA Development Kit (Peprotech, Rock Hill, NJ, USA), Human IL-4 Standard ABTS (PeproTech, Rock Hill, NJ, USA), Human IL-12 ELISA Development Kit (Peprotech, Rock Hill, NJ, USA), Human TNFα ELISA Development Kit (Peprotech, Rock Hill, NJ, USA), Human IFN-γ ELISA Development Kit (Peprotech, Rock Hill, NJ, USA), and Human Standard ABTS ELISA Development Kit (Peprotech), respectively. The ELISA analysis was performed using high binding ELISA plates (Greiner BioOne) at RT according to the manufacturer’s instructions. Optical density was measured using photospectrometer Spectramax 340 PC (Molecular Devices) at the wavelength 450 nm.
The represented data show values of two independent analyses normalized to the levels of cytokine expression for cells grown without GAIN scaffolds.

Conditioned medium was collected and centrifuged to remove cell debris, and shedded syndecan-4 as well as secreted CCL3, VEGF and CCL2 were detected according to the manufacturer’s instructions using the following ELISA kits: Human Syndecan-4 Assay Kit (JP27188, IBL), Human CCL3/MIP-1 alpha DuoSet (DY270-05, R&D), Human VEGF Quantikine ELISA Kit (DVE00, R&D) and Human CCL2/MCP-1 DuoSet, (DY279-05, R&D).