Milliplex madpk 71k adipokine kit

Glucose, triglycerides and non-esterified free fatty acids were assayed by enzymatic colorimetric tests (GOD-PAP, LPL/GOP-Trinder, Roche Diagnostics, Basil, Switzerland and HR Series NEFA-HR, Wako Diagnostics, Neuss, Germany). Plasma levels of insulin were measured using a Milliplex MADPK-71K adipokine kit (Merck-Millipore, Burlington, MA, USA) according to the manufacturer’s instructions. For estimation of insulin resistance, HOMA index was calculated as HOMA-IR = (FPG × FPI)/2430, where FPI was in microunits per milliliter and FPG in milligram per deciliter, as previously described [35 (link)].

Approximately 5 mg of each sample of the PFC (n = 5/group) was homogenized in 100 μl of homogenization buffer (0.05% Tween 20 and protease inhibitor cocktail (Thermo Fisher Scientific Inc., Waltham, MA, USA) in PBS, pH 7.2). After 3 cycles of 1 min and 50 Hz in the tissuelyzer (Quiagen, Germantown, Maryland, USA), samples were centrifuged at 11000×g for 30 min at 4 °C. After centrifugation, supernatants were transferred to a new tube and stored at −80 °C until the assay. Total protein content of each sample was measured using the BCA protein assay kit (Thermo Fisher Scientific Inc., Waltham, MA, USA). Levels of tumor necrosis factor-α (TNF-α), interleukin 1β (IL-1β), interleukin 6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), interleukin 4 (IL-4), and interleukin 10 (IL-10) were measured by X-Map technology using a Milliplex MADPK-71K adipokine kit according to the manufacturer’s description (Merck Millipore, Spain).