Ham s f 12 nutrient mixture dmem f12

Met, SAM, SAH, MTA, Hcy, Cysta, Cys, GSH, 5-MTHF, VB6, VB9, VB12, LC-MS-grade acetonitrile (ACN), LC-grade methanol (MeOH), formic acid, ascorbic acid, sodium hydroxide (NaOH) 0.1 M, hydrochloric acid (HCl), ammonium acetate (NH4OAc), tris-(2-carboxyethyl)phosphine (TCEP), L-Met, and DL-Met were purchased from Sigma-Aldrich (St. Louis, MO, USA). DL-HMTBA was purchased from Macklin (Shanghai, China). DL-MM was purchased from Taopu (Shanghai, China). Homocystine-d8 was purchased from CDN Isotopes (Pointe-Claire, Quebec, Canada). Ultrapure water was obtained from a Millipore-Q water system (Millipore, Bedford, MA, USA). Dulbecco's modified Eagle's medium and Ham's F-12 nutrient mixture (DMEM/F12), RPMI-1640 medium, fetal bovine serum (FBS), and penicillin–streptomycin were purchased from Gibco (Shanghai, China).

Four healthy male cynomolgus monkeys, aged 3 to 4 years and weighing about 3.5 kg, were purchased from Guangdong Chunsheng Biotechnology Development Co., Ltd. Monkeys were killed by intravenous overdose of sodium pentobarbital. Eyes were then nucleated and stored at 4°C. CM cells and TM cells were prepared as previously described [11 (link)]. The medium was composed of Dulbecco's modified Eagle's medium and Ham's F12 nutrient mixture (DMEM/F12) (Gibco, USA), supplemented with 20% fetal bovine serum (FBS) (Gibco, USA), 1% penicillin-streptomycin (Hyclone, USA), and 1 ng/mL recombinant human basic fibroblast growth factor (bFGF) (Gibco, USA). The cultures were incubated in a 37°C humidified incubator with an atmosphere of 20% O₂ and 5% CO₂. The medium was changed every 3 or 4 days. After the primarily cultured cells reached confluence, subsequent passages were performed. The confluent fourth to sixth passaged cells were used in the subsequent study.

Neonatal females were sacrificed by cervical dislocation on the designated time. Mouse ovaries were separated by microdissection in cold phosphate buffered saline (PBS) under a stereomicroscope (ZSA302, COIC, China) in sterile conditions. The isolated ovaries were cultured on an insert (PICM0RG50, Millipore, USA) in 6-well culture dishes (NEST, China) in 1200 μL Dulbecco’s modified Eagle’s medium/Ham’s F12 nutrient mixture (DMEM/F12) (GIBCO, Life Technologies, USA) plus ITS (1:100, Sigma, USA) and Penicillin-Streptomycin Solution at 37 °C, 5% CO2 and saturated humidity. Ovaries were cultured for 2 or 5 days to assess the role of CDC42, in either medium alone or medium supplemented with ML141 (5uM, Selleck, China) or ZCL278 (50uM, Selleck, China). ML141 is a selective reversible CDC42 inhibitor. ZCL278 is a selective CDC42 inhibitor.

The IPEC-J2 cell line used in this study was derived from jejunal epithelia of a neonatal piglet. It is a nontransformed cell line that in some respects mimics in vivo conditions when cultured on membrane inserts. Cells form a differentiated layer and are attached to each other via tight junctions apically. IPEC-J2 cells were seeded at a density of 1.5 × 10⁵ per well on six-well plates with Transwell polyester membrane inserts (pore size 0.4 μm; surface area 4.67 cm²; Sigma, Germany) coated with rat tail collagen (Sigma) in a 1.5 mL apical and 2.6 mL basolateral volume. Cells were maintained in complete medium containing 1 : 1 mixture of Dulbecco's Modified Eagle's Medium and Ham's F-12 Nutrient Mixture (DMEM/F12) supplemented with 5% FBS, 5 μg/mL insulin, 5 μg/mL transferrin, 5 ng/mL selenium, 5 ng/mL epidermal growth factor, and 1% penicillin-streptomycin (all from Fisher Scientific, USA). Cell cultures were tested by PCR and were found to be free of mycoplasma contamination. Cells were allowed to adhere for 24 h before being washed and refed every other day until confluence. They were grown at 37°C in a humidified atmosphere of 5% CO₂.

The IPEC-J2 cell line (derived from jejunal epithelia of a neonatal piglet) was kindly provided by Dr. Jody Gookin and Dr. Stephen Stauffer, Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC, USA and it was used between passage 50 and 75. IPEC-J2 cells were seeded at a density of 1.5x10⁵ per well on six-well plates with Transwell polyester membrane inserts (pore size 0.4 μm, Sigma-Aldrich, St. Louis, MO) coated with rat tail collagen (Sigma-Aldrich, St. Louis, MO) in a 1.5 ml apical and 2.6 ml basolateral volume. The surface area of the membrane insert was 4.67 cm². Cells were maintained in complete medium containing a 1:1 mixture of Dulbecco's Modified Eagle's Medium and Ham's F-12 Nutrient Mixture (DMEM/F12) supplemented with 5% FBS, 5 μg/ml insulin, 5 μg/ml transferrin, 5 ng/ml selenium, 5 ng/ml epidermal growth factor and 1% penicillin-streptomycin (all from Fisher Scientific, USA). Cell cultures were tested by PCR and were found to be free of mycoplasma contamination. Cells were allowed to adhere for 24 h before being washed and re-fed every other day until confluence. They were grown at 37°C in a humidified atmosphere of 5% CO₂.