Quantikine elisa human il 6 immunoassay

To quantify the secretion of pro- (IL-6, MCP-1, chemerin, and TNFα) and anti-inflammatory (adiponectin) adipokines, cells were treated with bioactive compounds for 48 h and conditioned medium was collected for adipokine quantification by ELISA according to the manufacturer’s instructions. The samples were diluted 5-60 fold for IL-6 detection (Quantikine ELISA Human IL-6 Immunoassay; R&D Systems, Minneapolis, MN, Intra-assay precision: CV <5 %, Inter-assay precision: CV <4 %), 3-10 fold for MCP-1 (Quantikine ELISA Human CCL3/MCP-1 Immunoassay; R&D Systems, Intra- and Inter-assay precision: CV <6 %) and 2-10 fold for chemerin (Quantikine ELISA Human Chemerin Immunoassay; R&D Systems, Intra-assay precision: CV ≤3 %, Inter-assay precision: CV <6 %) to match the standard curve. Adiponectin (Mercodia Adiponectin ELISA; Mercodia AB, Uppsala, Sweden, Intra-assay precision: CV <5 %, Inter-assay precision: CV <8 %) and TNFα (Quantiglo ELISA Human TNFα Immunoassay; R&D Systems, Intra-assay precision: CV <6 %, Inter-assay precision: CV <9 %) secretion were measured in undiluted samples. Levels of TNFα were detectable in adipocyte cultures treated with AC from only two subjects.

An enzyme-linked immunosorbent assay (ELISA) was used to determine IL-6 and MCP-1 protein levels. The cells were collected in ice-cold PBS and sonicated for 2 × 10 s on ice. The homogenate was centrifuged at 1700×g for 5 min at 4 °C and the supernatants collected. The levels of IL-6 and MCP-1 were measured in the supernatants using Quantikine ELISA Human IL-6 Immunoassay (R&D systems, #D6050, Minneapolis, MN, USA) and Quantikine ELISA Human MCP-1 Immunoassay (R&D systems, #DCP00), according to manufacturer’s protocol. IL-6 and MCP-1 concentrations were normalized to the amount of total protein in each sample. The total protein concentration was determined using the Bio-Rad DC protein assay (Bio-Rad, Hercules, CA, USA), according to manufacturer´s instructions.