Dulbecco s modified eagle medium nutrient mixture f 12 dmem f 12

Catalpol (purity > 98:72%) was obtained from Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China). Fetal bovine serum (FBS), 0.05% trypsin, Dulbecco’s Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F-12), and penicillin/streptomycin solutions were purchased from Gibco, Invitrogen (Carlsbad, CA, USA). Dimethyl sulfoxide (DMSO), N-Acetylcysteine (NAC) and hydrogen peroxide (H₂O₂) were bought from Sigma-Aldrich (St. Louis, MO, USA). Phosphate-buffered saline (PBS) and 3-(4,5- dimethyl thiazol-2-yl-)-2,5-diphenyl tetrazolium bromide (MTT) were obtained from Beijing Solarbio Science and Technology Co. Ltd. (Beijing, China). ROS, MMP, LDH, TUNEL, Apoptosis and Cell Cycle detection kits were purchased from Beyotime Biotechnology (Shanghai, China).

Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (DMEM-F12), ampicillin/streptomycin, and phosphate-buffered saline (pH 7.4) were purchased from Gibco Invitrogen (Carlsbad, CA). Trypsin-EDTA (0.25%) and fetal bovine serum were obtained from HyClone Thermo Scientific (Logan, UT). Triton X-100 was acquired from Research Organics (Cleveland, OH). Lipopolysaccharides (LPS) from Salmonella enterica serotype typhimurium L7261, as well as croton oil, indomethacin, isorhamnetin standard, and formic acid solution HPLC grade were purchased from Sigma-Aldrich (St. Louis, MO). Chromatography grade water and methanol (VWR International LLC, West Chester, PA) were used for high-pressure liquid chromatograph equipped with a photodiode array detector (HPLC-PDA) and liquid chromatograph/mass selective detector time-of-flight (LC/MSD TOF) analysis.

Human origin strain mesenchymal stem cells was obtained from deciduous tooth as mentioned before. The pulp was pulled from the teeth and washed twice with PBS (Gibco Invitrogen; pH 7.4, Grand Island, NY, USA). For digestion, 1 mg/ml trypsin (TrypLE, Gibco Invitrogen) was used for 30 min at 37 °C. Digestion was terminated by adding 4 ml Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F-12; Gibco Invitrogen) supplemented with 15% fetal bovine serum (FBS; Gibco Invitrogen) followed by centrifugation.
The samples were cut into appropriate fragment sizes. The fragments were plated into three or four wells of a 24-well plate. Fragments were maintained under a humidified atmosphere of 5% CO₂ at 37 °C. After ten days, when the fragments had expelled DPSCs, the fragments were trypsinized using standard procedures at 80% confluence to expand the lineage.

The mammary gland tissues were surgically isolated from lactating Guanzhong dairy goats. The mammary tissues were trimmed of visible fat and connective tissues and washed with phosphate-buffered saline (PBS) several times until the solution became pellucid and devoid of milk. The mammary tissues were minced into about 1 mm³ cubes and were then implanted into the 35-mm petri dish and were incubated at 37°C with saturated humidity and 5% CO₂. After 6 h, 2 ml culture medium was added to the culture dish. The culture medium consisted of Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F-12) (Invitrogen Corporation, Waltham, Massachusetts, USA) supplemented with 10% fetal bovine serum (Invitrogen Corporation, Waltham, Massachusetts, USA) and 100 IU/ml penicillin, 100 μg/ml streptomycin (Invitrogen Corporation, Waltham, Massachusetts, USA). The medium was replaced with fresh medium every 48 h until the cells migrated out of the tissue. About 10 days later, the cells spread across the bottom of the dish were passaged by digestion with 0.25% trypsin/ethylenediaminetetraacetic acid (EDTA). The cells were reseeded in a new 35-mm petri dish at a density of 2 × 10⁵ cells/cm² and cultured at 37°C under 5% CO₂. In present experiments, cells passaged within 9 times were used.