Fetal serum

Catechin standard ((±)-catechin hydrate) and pure soybean lipoxygenase-1 were purchased from Sigma Chemical Co., St. Louis, MO. The standard enzyme contained 46.000 units/mg protein. Other chemicals used were all analytical grade. RPMI medium, fetal serum, penicillin, streptomycin, L-glutamine, Triton, Hanks salt containing MTT ((3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide), and DMSO (dimethyl sulfoxide) were from Sigma, Cluj-Napoca, Romania.

The ICP1 cells were induced to differentiate by using the culture medium with 15% fetal serum and 0.2% oleic acid (Sigma, St Louis, CA, United States) after 12-h transfection. After 48-h induction, oil red O staining was performed by using an Oil Red Staining kit (Solarbio, Beijing, China) according to its protocol. The stained cells were captured in an electric microscope (Nikon, Tokyo, Japan). The oil red dye was extracted from the cells by using isopropanol solution. At last, the dye was quantified by a microreader (Biotek, Winooski, VT, United States).

The transfected cells were induced to differentiate into adipocytes by the culture medium with 15% fetal serum and 0.2% oleic acid (Sigma, CA, United States). The cells were washed with PBS and then, 4% formaldehyde was added to fix the cells for 30 min. Next, the cells were washed with PBS, followed by wash with 60% isopropanol, and finally stained with oil red O reagent for 30 min. The cells were then treated with 60% isopropanol for 15 s and rinsed with PBS 3 times for 3 min each time. The stained cells were observed, and images were captured under an electron microscope (Nikon, Tokyo, Japan). Oil red O dye was extracted from the cells in isopropanol solution and quantified in a microreader (Bio-Rad, CA, United States) at 510 nm. Cells were collected and resuspended in a 1.5 mL tube with 1 mL PBS. Finally, cells were counted in a cell counter (CountStar, Shanghai, China).