Elisa kits dou set

Cell supernatants were collected for evaluation of secreted cytokines. SFs (10⁴) were seeded in 200 μl of complete DMEM in 96-well plates pre-coated with bovine fibronectin (R&D, UK). For siRNA silencing experiments, the first siRNA transfection was performed in 12-well plates and when the first transfection was completed, the cells were detached for a second transfection and re-seeded in 96-well plates. Cells were cultured in the absence or presence of 10 ng/ml IL-1β for 24 hours, then supernatants were collected and frozen. ELISA kits (Dou set, R&D) were used to evaluate the levels of IL-6, MMP3 and CCL2 in the supernatants according to the manufacturer’s instructions. Cell viability was checked using a colorimetric MTS assay (abcam, catalog number ab223881).

Venous blood samples were drawn after an overnight fast. A 2-h 75 g oral glucose tolerance test (OGTT) was performed in follow-up individuals. The concentrations of plasma glucose (glucose oxidize method), TG, TC, HDL-C and low-density lipoprotein cholesterol (LDL-C) were assayed by the Hitachi 7060 C automatic biochemistry analysis system. HbA1c was assayed using the TOSOH G7 automatic analysis system. Insulin, adiponectin and leptin were measured by monoclonal antibody based enzyme-linked immunosorbent assay (ELISA) [29 ], which was developed in the Key Laboratory of Endocrinology, Peking Union Medical College Hospital. Insulin assay had an inter-assay coefficient of variation (CV) of < 9.0% and no cross-reactivity to proinsulin (< 0.05%). The intra- and inter-assay CVs were < 5.4 and < 8.5% for adiponectin, and < 7.4 and < 9.3% for leptin, respectively [30 (link)]. RBP4 was measured by ELISA kits (Dou set, R&D Systems, Minneapolis, MN, USA) with intra- and inter-assay CVs of 6.2 and 8.5%, respectively. Insulin resistance index was calculated by homeostasis model assessment of insulin resistance (HOMA-IR), HOMA-IR = fasting insulin (mU/L) × FG (mmol/L)/22.5 [31 (link)] or insulin sensitive index (Matsuda Index) (ISIM), ISIM = 10,000/(FG × fasting insulin) × (G × I), where G = mean serum glucose, and I = mean serum insulin concentration [32 (link)].