Free fatty acid assay kit

Serum was isolated from peripheral blood by centrifugation and stored at −80 °C until analysis. Cell supernatant was collected after the cells were cultured with basal condition or with isoprenaline stimulation for 24 h and immediately analyzed. Adipose tissue was freshly collected and processed immediately as previously described [36 (link)]. A Free Fatty Acid Assay Kit (Nanjing Jiancheng, A042-2) and a Glycerol Assay Kit (Nanjing Jiancheng, F005-1) were used to measure FFA and glycerol levels according to the manufacturer’s instructions.

The free fatty acid assay kit (Jiancheng, Nanjing, China) was used to detect the level of FFA according to the manufacture's instruction. 2 × 10⁶ cells were collected and homogenized in 200 μl chloroform/Triton X-100. In the assay, fatty acids can be converted to coenzyme A and oxidized, leading to the formation of optical color which is captured by measuring the absorbance at 546 nm with a microplate reader. Palmitic acid is used to generate a standard curve. Relative concentration of FFA in the test samples was calculated and normalized. Independent experiments were done in triplicate.

Levels of transforming growth factor-β₁ (TGF-β₁), interferon-γ (IFN-γ), interleukin-12 (IL-12), and tumor necrosis factor-α (TNF-α) in cell culture supernatant and serum were assessed with the ELISA assay (Proteintech, China). Free fatty acid was examined with the free fatty acid assay kit (#A042-1-1, Nanjing Jiancheng Bioengineering Institute, China), glucose uptake was examined by the glucose oxidase-peroxidase method with test kit (#361510, Rongsheng, China).