2.12. Adipokine measurements

Isolated adipocytes (~8 × 10⁵/cells) were incubated in a six‐well culture plate in Dulbecco's modified Eagle's medium containing 10% calf serum and 1% penicillin/streptozotocin at 37°C under humid atmosphere of 5% CO₂ for 30 hr. The final concentrations of the adipokines adiponectin, IL‐10, IL‐6, MCP‐1, resistin, and TNF‐α from the culture supernatant were determined using specific commercially available DuoSet ELISA kits in accordance with the manufacturer's (R&D Systems; catalogue numbers DY1119, DY417, DY406, DY479, DY1069 and DY 410, respectively). The concentrations of the cytokines are expressed in ng per 10⁶ cells as indicated. A similar procedure was used in our previous studies (de Sá et al., 2016).