Adipogenesis kit

Eight-ten-weeks old male and female mice were used. 20 Aox4^−/− and 20 WT were randomized and divided in two groups. 10 Aox4^−/− and 10 WT mice were fed standard chow (ND) while 10 Aox4^−/− and 10 WT animals were fed with a high fat diet (HFD, Teklad TD.88137). Food intake and body weight were measured twice a week. At the end of experiments, animals were sacrificed and blood, HG, adipose tissues as well as liver were isolated. Serum glucose, NEFA (non-esterified fatty acids), triglycerides and cholesterol were measured according to standard methods routinely used for human clinical samples. Total triglyceride content in tissues was measured using Adipogenesis Kit (Sigma-Aldrich).

The ‘Adipogenesis Kit’ (Sigma Aldrich) was used to detect the total cellular concentrations of triglycerides according to manufacturer’s manual. The coupled enzyme assay results in a colorimetric product, proportional to the trigylcerides present at day 8 of adipogenesis. Analyses were performed with the Synergy™ Mx microplate reader (BioTeK Inc., Winooski, VT, USA). Data were normalized to the protein concentration of each individual sample. Protein was isolated using radioimmunoprecipitation assay (RIPA) buffer and concentrations were determined by the BioRad Protein Assay (BioRad).

Liver sample 1 was thawed and the volume adjusted by methanol/water (1/1, v/v) to yield a tissue concentration of 50 mg/mL. The sample was then homogenized and metabolites and lipids were extracted as previously described in detail (Want et al. 2013 (link)). TAGs were analyzed utilizing a commercial kit (Sigma-Aldrich, Schelldorf, Germany, adipogenesis kit) from organic extract dried under a stream of nitrogen. Plasma insulin levels were determined by a mouse insulin ELISA (Mercodia, Sweden).

The Adipogenesis Kit (Sigma Aldrich) was used to detect total cellular concentrations of triglycerides by a coupled enzyme assay, which results in a colorimetric product, proportional to the trigylcerides present at day 8. The procedure followed the manufacturer’s instructions.

Serum samples were collected from the heart and separated by centrifugation at 1000 g for 10 min at 4 °C. Hitachi high-technologies global 7020 clinical analyzer was used to test serum biochemical parameters. The concentrations of triglycerides (TGs), cholesterol (CHOL), high-density lipoprotein (HDL), low-density lipoprotein (LDL), glucose, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured by DiaSys diagnostic systems (Shanghai, 200120, China). The concentration of plasma lipopolysaccharide (LPS) was measured using a Genscript ToxinSensorTM Chromogenic LAL Endotoxin Assay Kit (Jiangsu, China). The concentration of TGs in liver tissue was measured by using an Adipogenesis Kit (Sigma, 14508, USA, MAK040).