Dmem f12

Immortalized human endometrial stromal cells (hESC) were purchased from the American Type Culture Collection (ATCC CRL-4003TM) and cultured according to the manufacturer’s instructions^{73 (link)}. Briefly, stromal cells were cultured in DMEM/F12 (Sigma) supplemented with 10% charcoal-stripped FBS (CS-FBS, Biological Industries) at 37 °C in a humidified chamber with 5% CO2. To induce decidualization in vitro, stromal cells were treated with 1 μM Medroxyprogesterone 17-acetate (MPA, Sigma) and 0.5 mM dibutyryl cAMP (db-cAMP, Sigma) in DMEM/F12 with 2% CS-FBS for 6 days. The medium was changed every 48 h. Under in vitro decidualization, stromal cells were treated with 0.032, 0.16, 0.8, 4, and 20 μM P (Sigma) for further analysis, respectively. The highest treatment dose of P has no significant toxic effect on cell viability.

The hSCAPs were seeded on poly-d-lysine (Sigma-Aldrich) coated cover slips (Electron Microscopy Sciences, Hatfield, PA, USA) in 6-well plates at a density 1 × 10⁵ cells/well and pre-incubated with the optimal condition of resveratrol (RSV-hSCAPs) or without resveratrol (hSCAPs). Then, both hSCAPs and RSV-hSCAPs were exposed to 2 phases of neuronal induction medium. First, the cells were incubated with Dulbecco’s Modified Eagle Medium: Nutrient Mixture F-12 (Ham) (DMEM/F-12, Gibco, Life Technologies) supplemented with 10% FBS, 100 U/mL penicillin, 100 μM/mL streptomycin, 10 ng/mL basic fibroblast growth factor (bFGF, Gibco Life Technologies), and 500 μM β-mercaptoethanol (Sigma-Aldrich) for 24 h. After that, the cells were induced into a phase II neuronal induction medium which consisted of DMEM/F-12, 100 U/mL penicillin, 100 μM/mL streptomycin, 2% DMSO, and 100 μM butylated hydroxyanisole (BHA, Sigma-Aldrich) for 6 h. The negative control hSCAPs (crt-hSCAPs) was pre-incubated for 12 h with αMEM, 100 U/mL penicillin, 100 μM/mL streptomycin, and then cultured with DMEM/F-12, 10% FBS, 100 U/mL penicillin, and 100 μM/mL streptomycin for 24 h. The medium was then replaced with DMEM/F-12, 100 U/mL penicillin, and 100 μM/mL streptomycin for 6 h.

Mouse endometrial stromal cells were isolated and treated as previously described [43 (link)]. In brief, mouse uteri on day 4 were split longitudinally and digested with 6 mg/mL dispase II (Roche Applied Science, Indianapolis, IN) and 1% trypsin (Amresco, Cleveland, OH) in HBSS (Sigma-Aldrich). After luminal epithelial cells were removed by rinsing, the remaining uteri were digested with 0.15 mg/mL collagenase I (Invitrogen, Houston, TX, USA). The collected stromal cells were cultured with DMEM/F12 (Sigma-Aldrich) containing 10% charcoal-treated FBS (cFBS; Biological Industries, Cromwell, Israel). To induce in vitro decidualization, mouse stromal cells were treated with 1 mM progesterone (Sigma-Aldrich) and 10 nM 17β-estradiol (Sigma-Aldrich) in DMEM/F12 (Sigma-Aldrich) containing 2% cFBS (Biological Industries).

Murine chondrogenic ATDC5 cells (Sigma-Aldrich; 99072806) were cultured in DMEM:F12 (Gibco, Billings, MT, USA; 11320-033) with 5% FCS (Gibco; 10437-028) and 100 U/mL penicillin and streptomycin (Gibco; 15140122) at 37 °C, 5% CO2 and 95% relative humidity.
Primary murine chondrocytes were isolated from rib cage cartilage of 10-day-old mice by collagenase (Sigma-Aldrich; C9891) digestion in DMEM:F12 and cultured in DMEM:F12 with 10% FCS and 50 μg/mL ascorbic acid at 37 °C, 5% CO₂ 5% O₂ and 95% relative humidity.

HepG2, SW872, SGBS, 293T, 3T3L1, Huh-7, and MIN6 cell lines were maintained at 37° with 5% CO_2. HepG2 cells (ATCC, HB-8065) were cultured in MEM-α (Gibco) supplemented with 10% FBS and 1 mM sodium pyruvate. SW872 cells (ATCC, HTB92) were cultured in DMEM:F12 (Sigma) supplemented with 10% FBS. SGBS cells (Wabitsch et al. 2001 (link)) were generously provided by Dr. Martin Wabitsch (University of Ulm) and cultured in DMEM:F12 (Sigma) supplemented with 10% FBS and 5% 3.3 mM biotin/1.7 mM panthotenate solution. SW872 and SGBS cells were transfected in the undifferentiated, preadipocyte state. 293T cells (ATCC, CRL-3216) were cultured in DMEM (Sigma) supplemented with 10% FBS and 200 mM L-glutamine. 3T3-L1 cells (ATCC, CL-173) were cultured and differentiated as described in the ATCC protocol. Huh-7 cells (JCRB0403, Japanese Collection of Research Bioresources Cell Bank, National Institute of Biomedical Innovation), were cultured in DMEM with high glucose (Gibco) with 10% FBS, 1 mM sodium pyruvate, 1 mM nonessential amino acids (Sigma), and 1 mM L-glutamine. MIN6 cells (Miyazaki et al. 1990 (link)) were cultured in DMEM (Sigma), supplemented with 10% FBS, 1 mM sodium pyruvate, and 0.1 mM β-mercaptoethanol.