Stemsure monothioglycerol solution

mESCs were cultured in mESC medium (DMEM high Glucose (Nacalai Tesque, Kyoto, Japan) supplemented with 15% Fetal Bovine Serum (FBS) (Thermo Fisher Scientific, Waltham, MA), 100 mM Non-Essential Amino Acids (Nacalai Tesque, Kyoto, Japan), 0.5 mM StemSure Monothioglycerol Solution (FUJIFILM Wako Pure Chemical, Osaka, Japan), 1000 U/mL LIF (FUJIFILM Wako Pure Chemical, Osaka, Japan), and 100 U/mL Penicillin/Streptomycin solution (Nacalai Tesque, Kyoto, Japan)). To form EBs, mESCs were cultured in an EB differentiation medium (DMEM high Glucose supplemented with 20% FBS, 100 mM Non-Essential Amino Acids, 0.5 mM StemSure Monothioglycerol Solution, and 100 U/mL Penicillin/Streptomycin solution). NIH3T3 cells, HEK293T cells, and MEFs were cultured in DMEM medium (DMEM high Glucose supplemented with 10% FBS and 100 U/mL Penicillin/Streptomycin solution). mESCs were allowed to differentiate by culturing the cells in the DMEM medium with 5 µM of retinoic acid (Sigma-Aldrich, St. Louis, MO, USA).

NIH3T3 cells were cultured in DMEM [DMEM high Glucose (Nacalai Tesque) supplemented with 10% Fetal Bovine Serum (FBS; Nissui) and 100 U/mL Penicillin/Streptomycin solution (Nacalai Tesque)]. Mouse embryonic stem cells (mESCs) EB5 were cultured in mESC medium [DMEM high Glucose supplemented with 15% FBS, 100 mM Non-Essential Amino Acids (Nacalai Tesque), 0.5 mM StemSure Monothioglycerol Solution (Wako), 1,000 U/mL Leukemia Inhibitory Factor (LIF; Wako), and 100 U/mL Penicillin/Streptomycin solution]. For NSC differentiation, mESCs were cultured in NSC medium [DMEM/F12 (Nacalai Tesque) supplemented with B27 supplement (Wako), 20 ng/mL basic-FGF (R&D system), 20 ng/mL EGF (PeproTech), 0.005% Bovine Serum Albumin (BSA; Wako), 5 µg/mL Heparin (Nacalai), 100 U/mL Penicillin/Streptomycin solution, and 50 µg/mL Ascorbic acid (Nacalai Tesque)]. NSCs were maintained in a plate coated with 5 µg/mL Laminin (Wako). For astrocyte differentiation, NSCs were cultured in astrocyte differentiation medium (DMEM/F12 supplemented with 1% FBS, B27 supplement, 0.5 mM StemSure Monothioglycerol Solution, 0.005% BSA, and 100 U/mL Penicillin/Streptomycin solution). Protein expression levels were manipulated by addition of Shield1 (TaKaRa Bio) or 5-Ph-IAA (BioAcademia).