Mouse egf

Zebrafish liver cell line ZFL (Ghosh et al. 1994 (link); Eide et al. 2014 (link)) (CVCL_3276) was cultured in a medium consisting of 50% (v/v) Leibovitz’s L-15 (Sigma-Aldrich, Steinheim, Germany), 35% (v/v) Dulbecco’s modified Eagle's medium (Gibco, Paisley, UK), 15% (v/v) Ham’s Nutrient Mixture F-12 (Gibco, Paisley, UK), and phenol red. Additionally, 150 mg/L sodium bicarbonate (Gibco, Paisley, UK), 15 mM HEPES (Gibco, Paisley, UK), 10 µg/mL bovine insulin (Sigma-Aldrich, Steinheim, Germany), 50 ng/mL mouse EGF (Sigma-Aldrich, Steinheim, Germany), and 5% (v/v) fetal bovine serum (Gibco, Paisley, UK) were supplemented. The cells were cultured in a humidified environment at 28 °C and atmospheric CO₂. The cells were passaged weekly in a 1:20 ratio, using PBS (pH 7.4) (Medicago, Uppsala, Sweden) for washing and 0.25% (w/v) trypsin-EDTA (Sigma-Aldrich, Steinheim, Germany) for detachment.

Cells collected by the cell sorter were cultured in non-adherent 24 well plates (Corning, New York, NY, USA) (LepR/Tom⁺ BM cells: 4781‒9849 cells/well, LepR/Tom⁻ BM cells: 18,642‒201,149 cells/well, LepR/Tom⁺ PDL cells: 83‒348 cells/well, LepR/Tom⁻ PDL cells: 9125‒61,003 cells/well) with spheroid-forming media^{22 (link),27 (link)–29 (link)} (1:2 ratio of DMEM/F-12 (1:1) (21331-020) and Human Endothelial Medium (11111-044) supplemented with 3.75% Chicken Extract, 0.1 mM β-ME, 1% Non-essential amino acids, 1% Antibiotic–Antimycotic, 1% N2, 2% B27, 20 ng/mL mouse PDGF-AA (all from Thermo Fisher Scientific), 20 ng/mL human bFGF (ReproCELL Inc., Kanagawa, Japan), 20 ng/mL mouse oncostatin M, 20 ng/mL mouse IGF-1 (all from R&D SYSTEMS), 20 ng/mL mouse EGF (Sigma-Aldrich, St. Louis, MO, USA)). After culturing for 14 days, spheroid-forming efficiency was determined. Fluorescence and phase-contrast images of spheroids were acquired using an All-in-One Fluorescence Microscope (BZ-X700) equipped with a BZ-X-Viewer, BZ-X Analyzer (all from KEYENCE, Osaka, Japan), a CFI Plan Fluor DL (4×/0.13), and a CFI Plan Fluor DL (10×/0.45) (both from Nikon, Tokyo, Japan).

The zebrafish liver cell line (CVCL_3276) (Ghosh et al. 1994 (link)) was purchased from ATCC (Mannassas, USA). A nutrition medium consisting of 50% (v/v) Leibovitz’s L-15 (Sigma-Aldrich, Steinheim, Germany), 35% (v/v) Dulbecco’s modified Eagle’s medium (Gibco, Paisley, UK), 15% (v/v) Ham’s Nutrient Mixture F-12 (Gibco, Paisley, UK), and phenol red, supplemented by 150 mg/L sodium bicarbonate (Gibco, Paisley, UK), 15 mM HEPES (Gibco, Paisley, UK), 10 μg/mL bovine insulin (Sigma-Aldrich, Steinheim, Germany), 50 ng/mL mouse EGF (Sigma-Aldrich, Steinheim, Germany), and 5% (v/v) fetal bovine serum (Gibco, Paisley, UK), was used for cultivation. The cells were cultured in a humidified environment at 28 °C and atmospheric CO₂. Further, cells were passaged weekly in a 1:20 subcultivation ratio. Phosphate buffered saline (PBS, pH 7.4; Medicago, Uppsala, Sweden) was used for washing and 0.25% (w/v) trypsin–EDTA (Sigma-Aldrich, Steinheim, Germany) for cell detachment. All experiments were conducted within passage numbers 5 to 32.

mCCD_cl1 cells were maintained at 37 °C and 5% CO₂ in DMEM/F-12 medium (Thermofisher, 31331093, Waltham, MA, USA), supplemented with 5 µg/mL insulin (Sigma, I-1882, St. Louis, MO, USA), 50 nM dexamethasone (Sigma, D-8893), 60 nM selenium (Sigma, S-9133), 5 µg/mL transferrin (Sigma, T-1428), 1 nM triiodothyronine (Sigma, T-5516), 5 ng/mL mouse EGF (Sigma, E-4127), 100 µg/mL penicillin/streptomycin glutamine (Thermofisher, 10378016), and 2% decomplemented fetal bovine serum (Thermofisher, 10270106). For hypoxic culture, the medium was freshly replaced, and cells were maintained for the indicated time at 0.2% O₂ and 5% CO₂ in a gas-controlled glove box (InvivO2 400, Baker Ruskinn, Bridgend, UK). Cellular viability was routinely analyzed by trypan blue exclusion using the TC20 automated cell counter (Biorad, Hercules, CA, USA).

All protocols performed were approved by the Animal Care and Use Committee of Nanjing Agriculture University and were performed in accordance with Animal Research Institute Committee guidelines. Porcine ovaries were collected from the local slaughterhouse and then transported to laboratory within 3 h in sterile saline (0.9% NaCl) containing 0.03 g/mL of penicillin and 0.03 g/mL of streptomycin at 37°C. Cumulus-oocyte complexes (COCs) were extracted from 3 to 6 mm follicles of ovaries by aspirating with a 20-gauge needle attached to a 5-ml disposable syringe. Oocytes with compact cumulus cells and a uniform ooplasm were selected for in vitro maturation (IVM). The COCs was washed three times with IVM medium [TCM199 (St. Louis, MO, United States,# M2154) supplemented with 75 μg/ml of penicillin, 50 μg/ml of streptomycin, 0.5 μg/ml of LH, 0.5 μg/ml of FSH, 10 ng/ml of epidermal growth factor (mouse EGF, Sigma, #E4127), and 0.57 mM cysteine] and cultured in each well of a four-well dish (Nunc, Roskilde, Denmark) containing 500 μl of IVM medium covered with 200 μl mineral oil at 38.5°C in a humidified atmosphere of 5% CO₂ incubator for IVM. After 42–46 h cultivation, COCs were transferred to 0.1% hyaluronidase (w/v) for 3 min at 38.5°C. After three to four rinses with TCM199, matured MII oocytes were collected for next treatment.

Zebrafish liver cell line ZFL^{26 (link),27 (link)} (CVCL_3276) was cultured in a medium consisting of 50% Leibovitz’s L-15 (Sigma-Aldrich, Steinheim, Germany), 35% Dulbecco’s modified Eagle’s medium (Gibco, Paisley, UK), 15% Ham’s Nutrient Mixture F-12 (Gibco, Paisley, UK), and phenol red. Additionally, 150 mg/L sodium bicarbonate (Gibco, Paisley, UK), 15 mM HEPES (Gibco, Paisley, UK), 10 µg/mL bovine insulin (Sigma-Aldrich, Steinheim, Germany), 50 ng/mL mouse EGF (Sigma-Aldrich, Steinheim, Germany), and 5% fetal bovine serum (Gibco, Paisley, UK) were supplemented. The cells were cultured in a humidified environment at 28 °C and atmospheric CO₂. The cells were passaged every 3 to 4 days in a 1 to 4 ratio, using PBS (Medicago, Uppsala, Sweden) for washing and 0.25% Trypsin-EDTA (Sigma-Aldrich, Steinheim, Germany) for detachment.

HEK293T cells (ATCC, CRL-3216) and human newborn foreskin fibroblasts (HFFs, from ATCC, SCRC-1041) were purchased from ATCC. The SCR029 cells (Chemicon, SCR029) are well-characterized mouse cortical NSCs obtained from Chemicon. These cells were tested for mycoplasma contamination before experiments and they are negative. HEK293T cells, HFFs, and MEFs were all expanded in the MEF medium [Dulbecco’s modified Eagle’s medium (DMEM, Hyclone), 10% fetal bovine serum (FBS) (Gibco), 1 × Pen/Strep (Gibco), 1 × MEM non-essential amino acids (Gibco), and 0.008% (v/v) 2-mercaptoethanol (Sigma)]. All the NSCs were plated on culture dishes pre-coated with 5 μg/ml poly-l-ornithine (Sigma) and 5 μg/ml laminin (Sigma). miNSCs and human iNSCs were cultured in the NSC medium containing the N3 medium supplemented with 10 ng/ml recombinant mouse EGF (R&D systems) and 10 ng/ml recombinant human bFGF (R&D Systems), with or without 2 ng/ml Dox (Sigma). The N3 medium contains DMEM/F12 (Life Technologies) with 1 × Pen/Strep, 25 μg/ml insulin (Sigma), 50 μg/ml Apo-transferrin (Sigma), 1.28 ng/ml progesterone (Sigma), 16 ng/ml putrescine (Sigma), and 0.52 μg/ml sodium selenite (Sigma). The SCR029 cells were cultured in the Neural Stem Cell Expansion Medium (Chemicon, SCM003) supplemented with 20 ng/ml mouse EGF, 20 ng/ml human bFGF, and 2 μg/ml heparin (Sigma).