Enzychrom glycerol assay kit

Venous blood samples were collected by venipuncture at the indicated time points (Figure 1). Blood samples were collected into ethylenediaminetetraacetic acid (EDTA)-containing tubes. The EDTA-containing tubes for plasma separation were immediately centrifuged at 3000 rpm (approximately 1700× g) for 10 min at 4 °C. Plasma samples were stored at −80 °C until analysis. Plasma total protein, albumin, albumin/globin ratio (A/G), total bilirubin, aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), γ-glutamyl transpeptidase (γ-GTP), creatine kinase (CK), amylase, total cholesterol (T-CHO), low-density lipoprotein cholesterol (LDL-CHO), high-density lipoprotein cholesterol (HDL-CHO), aldolase, triglyceride, free fatty acid, uric acid, blood urea nitrogen, creatinine, glucose, bile acid, blood concentration of calcium ion, chloride ion, potassium ion, sodium ion, and inorganic phosphorus were measured by Koutou-Biken Co. (Tsukuba, Japan). Glycerol, lactate, and ketone body concentrations were measured with an EnzyChrom™ Glycerol Assay Kit, EnzyChrom™ Lactate Assay Kit, and EnzyChrom™ Ketone Body Assay Kit (Bioassay systems, Hayward, CA, USA).

The effect of the green bean extracts on lipolysis was quantified by adding increasing concentrations (20, 40, 60, 500, 800, and 1000 µg/ml) of green bean extract to differentiated adipocytes for 48 h. Isoproterenol (Sigma, USA, Lot SLBK3425V), a known stimulator of adipolysis, was used as a positive control in all assays. Glycerol release was quantified using a kit (EnzyChrom™ Glycerol Assay Kit, BioAssay Systems, USA).

SalB was purchased from Biopurify Phytochemicals Co., Ltd. (Cat. No. 15051803, Chengdu, China). Dulbecco’s modified Eagle’s medium (DMEM), Penicillin-streptomycin and 0.25%Trypsin-EDTA were purchased from Gibco (Cat. Nos. 1768707, 15140-122, and 1846496, Carlsbad, CA, United States). Fetal bovine serum (FBS) was purchased from Tianhang Biotechnology Co., Ltd. (Cat. No. HZ001, Hangzhou, China). Dexamethasone (DEX), insulin, and 3-isobutyl-1-methylxanthine (IBMX) were purchased from Sigma (Cat. No. I5500, d1756, and 17018, MO, United States). Oil Red O was purchased from Solarbio (Cat. No. G1260, Beijing, China). Glucose Oxidase Method kit was obtained from Applygen Technologies Inc. (Cat. No: E1010, Beijing, China). Enzychrom Glycerol Assay Kit was purchased from BioAssay Systems (Cat. No. EGLY-200, Hayward, CA, United States). GW9662 was bought from Selleck (Cat. No. S2915, Houston, TX, United States). Mito Tracker Green (MTG) was obtained from KeyGEN BioTECH (Cat. No. KGMP0072, Nanjing, China). The primers were designed and synthesized by Sangon Biotech (Shanghai, China). Antibody to PGC-1α was purchased from Proteintech (Cat. No. 20658-1-AP, Chicago, IL, United States). Fluorescein-Conjugated Goat anti-Rabbit lgG (H+L) was purchased from ZSGB-BIO (Cat. No. ZF0311, Beijing, China).