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2 protocols using mem non essential amino acid solution neaa

1

Efficient Neural Induction Protocol

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The cells were switched to the neural induction medium containing 1:1 mixture of N2/B27 medium containing 10 ng/ml basic fibroblast growth factor (b-FGF), 1 µM Dorsomorphin dihydrochloride (Tocris, 3093), 10 µM SB431542 (Tocris, 1614), 100 ng/ml mouse recombinant Sonic Hedgehog (SHH)-C25II (Genscript, Z03050-50), and 10 µM CHIR99021 (Sigma, SML1046). The N2 medium consisted of DMEM/F12 medium (Gibco) with 1× N2 supplement (Gibco, 17502048), 5 µg/ml insulin (Sigma, 19278), 1 mM L-Glutamine (Lonza, 17605E), 100 µM MEM-Non-Essential Amino Acid solution (NEAA) (Gibco), 100 µM 2-mercaptoethanol (Sigma, M3148), and 1:100 Penicillin-Streptomycin (Lonza, 17602E). The B27 medium consisted of a Neurobasal medium (Gibco) and 1× B27 supplement (Gibco, 17504044). The cells were washed daily using DPBS and maintained in the induction medium.
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2

Culturing MDA-MB-231, IGROV1, and UC-MSCs

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MDA-MB-231 human breast cancer cells and IGROV1 human ovarian cancer cells were cultured with DMEM (high glucose) medium (Corning, Lowell, MA) supplemented with 10% fetal bovine serum (FBS) (Corning, Lowell, MA) and 1% penicillin streptomycin solution (Gibco, Rockville, MD). Medium for MDA-MB-231 cells was also supplemented with 1% MEM nonessential amino acid solution (NEAA; Gibco). UC-MSCs were isolated as described before [17 (link), 18 (link)] and cultured with DMEM/F12 medium (Gibco) containing 10% FBS (Corning), 1% penicillin streptomycin solution (Gibco), and 10 ng/ml human recombinant epidermal growth factor (EGF; Gibco). All cell lines were maintained at 37°C in a 5% CO2 incubator. To be trackable in direct coculturing model, MDA-MB-231 cells were transduced with lentiviral vector carrying green fluorescence protein (GFP) and selected with blasticidin.
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