Dulbecco s modified eagle medium f12

Isolation and dissociation of stem cells from intestinal tumors of Min mice and normal epithelia from wild-type C57BL/6 mice were performed as described previously8 (link). Isolated tumor cells and epithelial cells were embedded in Matrigel on ice (growth factor-reduced, phenol red-free; BD Biosciences) and seeded in 48-well plates. The Matrigel was polymerized for 10 minutes at 37 °C, and overlaid with 250 μL/well basal culture medium (advanced Dulbecco’s modified Eagle medium/F12 supplemented with penicillin/streptomycin, 10 mmol/L HEPES, Glutamax, 1 × N2, 1 × B27 [all from Thermo Fisher Scientific], and 1 mmol/L N-acetylcysteine [Sigma-Aldrich]) containing the following optimized growth factor combinations: epidermal growth factor (EGF) and noggin for intestinal tumors; EGF, noggin and Rspo1 for intestinal epithelia. Intestinal tumor organoids were treated with 1 or 3 μM 5-Aza-CdR. Twenty-four hours after treatment, 5-Aza-CdR was removed from the culture medium, and regular culture medium was employed thereafter.

Isolation and dissociation of stem cells from normal intestinal epithelia of wild-type C57BL/6 mice were performed.^{3 (link)} Isolated intestinal epithelial stem cells were embedded in Matrigel (growth factor-reduced, phenol red-free; BD Biosciences) and seeded in 48-well plates. The cells were overlaid with 250 μL/well basal culture medium (advanced Dulbecco’s modified Eagle medium/F12 supplemented with penicillin/streptomycin, 10 mmol/L HEPES, Glutamax, 1 × N2, 1 × B27 [all from Thermo Fisher Scientific], and 1 mmol/L N-acetylcysteine [Sigma-Aldrich]) containing EGF, noggin, Y-27632, and Rspo1. For this study, we considered mice over 50-weeks-old to be aged mice, and considered establishment of intestinal epithelial organoids to be successful when they could be cultured for over 5 passages. Images were acquired using either a fluorescence microscope equipped with phase-contrast optics (CKX41, Olympus) or the Olympus Fluoview system (FV1000D, Olympus). Before observation, organoids were fixed with 4% paraformaldehyde phosphate buffer solution (Wako) for 30 min and 0.25% Triton X-100 (Sigma) for 15 min at room temperature to increase cellular permeability. DNA was stained with DAPI (Molecular Probes). All experiments and procedures were approved by the Keio University Animal Research Committee, and all methods were carried out in accordance with the approved guidelines.

The human neuroblastoma cell line SH‐SY5Y was cultured in Dulbecco's modified Eagle medium/F12 (Thermo Fisher Scientific) containing 10% foetal bovine serum at 37°C in a humidified atmosphere containing 95% air and 5% CO₂. Cells were rendered quiescent by serum starvation for 24 hours before all experiments. Hypoxic injury was induced by hypoxia for 6 hours (ie incubation in OGD medium containing 2.3 mmol/L CaCl₂, 5.6 mmol/L KCl, 154 mmol/L NaCl, 5 mmol/L Hepes, and 3.6 mmol/L NaHCO₃ [pH 7.4] and under an atmosphere of 5% CO₂, 95% N₂ and <0.1% O₂). The DMSO or HDAC inhibitor was treated with OGD medium. The neutralizing antibody of BDNF (1, 3 or 10 µg/mL) was treated for 24 hours before OGD condition.

Astrocyte thawing/centrifuging medium: Dulbecco’s Modified Eagle Medium/F12 (Thermo Fisher Scientific, Waltham, MA, USA).
Sensory Neuron Seeding Medium: Custom Senso-MM for co-cultures and Senso-MM for SN monocultures Reagents for seeding and maintaining sensory neuron/astrocyte co-culture were purchased from Anatomic (Minneapolis, MN, USA).

GT was purchased from the market in Taichung, Taiwan. IS (purity 97%) was obtained from Alfa Aesar (Lancaster, UK). 6,7-dimethoxycoumarin (6,7-DMC, purity 98%) was supplied by Aldrich Chemical Co. (Milwaukee, WI, U.S.A.). EC (purity 90%), ECG (purity 98%), EGCG (purity 95%), formic acid, probenecid, phosphoric acid (glacial, 85%) and methyl-paraben were purchased from Sigma Chemical Co. (St. Louis, MO, U.S.A.). EGC (purity 92.7%) was obtained from ChromaDex, Inc. (Irvine, CA, U.S.A.). and ethyl acetate were LC grade and obtained from ECHO Chemical Co. (Miaoli Hsien, Taiwan). Acetonitrile was LC/MS grade and was purchased from Millinckrodt Baker, Inc. (Phillipsburg, NJ, U.S.A.). Fetal bovine serum was obtained from Biological Industries Inc. (Kibbutz, Beit Haemek, Israel). Penicillin-Streptomycin-Glutamine, Dulbecco’ s Modified Eagle Medium, trypsin/EDTA, Hank’s Balanced Salt Solution and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid were purchased from Invitrogen (Carlsbad, CA, U.S.A.). Dulbecco’s Modified Eagle Medium F12 was obtained from Thermo Fisher Scientific Inc (Waltham, MA, U.S.A.). 6-Carboxyfluorescein was purchased from AAT Bioquest Inc. (Sunnyvale, CA, U.S.A.) and 5-carboxyfluorescein was obtained from Acros Organics (Geel, Belgium). Milli-Q plus water (Millipore, Bedford, MA, U.S.A.) was used for all preparations.