Adipogenesis assay kit

Mouse primary hepatocytes were plated in six-well plates in Dulbecco’s modified Eagle’s medium (DMEM) medium containing 10% FBS. Intracellular triglyceride (TG) contents were measured using an Adipogenesis Assay Kit (Sigma-Aldrich, Cat#MAK040) according to the manufacturer’s instructions and normalized to total protein concentrations. Intracellular TG levels were expressed in nmol/μg protein.

The effect of PP and ACN fractions on lipid content in hypertrophied adipocytes was determined by the Oil Red O (Sigma–Aldrich, Poznań, Poland) staining method described previously [13 (link)] and by total triglycerides (TG) measurement using the Adipogenesis Assay Kit (Sigma–Aldrich, Poznań, Poland) in accordance with the manufacturer’s instruction. Intracellular TG content was determined by an enzyme assay. A colorimetric product corresponding to the TG present was measured at 570 nm. TG concentration was calculated based on the curve plotted for the TG standard.

Total concentrations of triglycerides (TG) in differentiated 3T3-L1 adipocytes were determined using Adipogenesis Assay Kit (Sigma-Aldrich) according to the manufacturer’s protocol. Intracellular TG content was measured by a coupled enzyme assay, which resulted in a fluorometric product detected at λ_ex = 535 nm and λ_em = 587 nm (Tecan M200 Infinite), which was proportional to the TG present. The TG concentration was calculated based on the curve plotted for TG standards.

Baicalein was purchased from Sigma-Aldrich, USA. Phospholipon 90G (phosphatidylcholine 90%) was acquired from American Lecithin Company, Germany. All cell culture materials including, Dulbecco’s modified eagle’s medium (DMEM), fetal bovine serum (FBS), L-glutamine, adipocyte differentiation medium (ADM) were obtained from Gibco (Grand Island, NY, United States). Oil red o staining kit was purchased from Lifeline Cell Technology (Frederick, MD, United States). Adipogenesis assay kit was purchased from Sigma–Aldrich (St. Louis, MO, United States). All reagents and solvents are for research use only. Human foreskin fibroblasts (Hs68) were obtained from ATCC (Manassas, VA, United States) and Murine macrophages RAW 264.7 were purchased from Bioresource collection and Research Center, Food Industry Research and Development Institute, Taiwan. Both cell lines were cultured in DMEM supplemented with 10% v/v FBS, 100 units/ml penicillin and 100 μg/ml of streptomycine under steady state condition at 37°C with 5% CO₂ in a humidified incubator.

Quantitative analysis of triglyceride content in ADM-induced Hs68 fibroblasts was conducted using an adipogenesis assay kit (Sigma–Aldrich) according to the manufacturer’s instructions. Hs68 fibroblasts were cultured and adipogenesis was induced as described above. Free drug, empty liposomes or baicalein-loaded liposomes were added to cells respectively at day 7. After 21 days of incubation, 100 μl of lipid extraction buffer was added and incubated for 30 min at 90°C. Cell medium became cloudy and then cooled down at room temperature. Cell medium was shaken for 1 min for homogenization followed by transfering 5–50 μl of lipid to another 96 well plate which were filled with adipogenesis assay buffer up to a total volume of 50 μl. Lipase solution (2 μl) was added and incubated for 10 min at room temperature for the degradation of triglyceride, following by adding master reaction mix for reaction of 30 min before measuring the absorbance at 570 nm using the ELISA reader (Tecan, infinite M200). Triglyceride content in Hs68 fibroblasts was calculated from the triglyceride (triolein)-equivalent standard curve.