5G6GR mouse ES cells were used for total RNA extraction and scRNA-seq. 5G6GR mouse ES cells were provided by Hitoshi Niwa (Laboratory for Stem Cell Biology, Institute of Molecular Embryology and Genetics at Kumamoto University). This cell line was constructed by randomly incorporating the linearized Gata6-GR-IRES-Puro vector into EB5 ES cells46 (link). The cells were cultured in a feeder-free gelatin-coated dish in 10% fetal calf serum containing Glasgow minimal essential medium (Sigma-Aldrich), 1000 U/mL leukemia inhibitory factor (ESGRO; Millipore), 100 µmol/L 2-mercaptoethanol (Thermo Fisher), 1× non-essential amino acids (Thermo Fisher), 1 mmol/L sodium pyruvate (Thermo Fisher), 2 mmol/L l -glutamine (Sigma-Aldrich), 0.5× penicillin/streptomycin (Thermo Fisher), 0.5 µg/mL puromycin (Sigma-Aldrich), and 10 µg/mL blasticidin (Thermo Fisher). To assess PrE differentiation, 5G6GR ES cells were cultured in differentiation medium containing 100 mmol/L dexamethasone rather than blasticidin. The cells were cultured for 72 h.
Glasgow minimal essential medium
Manufactured by Merck Group
Sourced in United States, Germany
Glasgow minimal essential medium is a cell culture medium formulated to support the growth and maintenance of a variety of cell lines. It provides the necessary nutrients and components to sustain cell viability and proliferation in an in vitro environment.
Automatically generated - may contain errors
Lab products found in correlation
2 mercaptoethanol, by Merck Group (2 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Blasticidin, by Thermo Fisher Scientific (1 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (1 mentions)
2 mercaptoethanol, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Merck Group (1 mentions)
Puromycin, by Merck Group (1 mentions)
Knockout serum, by Merck Group (1 mentions)
Bsa fraction 5, by Thermo Fisher Scientific (1 mentions)
Pd0325901, by Fujifilm (1 mentions)
Chir99021, by Cayman Chemical (1 mentions)
Leukemia inhibitory factor, by Merck Group (1 mentions)
Non essential amino acids (neaa), by Harvard Bioscience (1 mentions)
L glutamine, by Harvard Bioscience (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
4 protocols using glasgow minimal essential medium
1
5G6GR mouse ES cells: scRNA-seq protocol
2
Neural Differentiation of Mouse and Human ESCs
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46C ESCs (expressing Sox1-GFP) derived from strain 129Ola mice were cultured on 0.1% gelatin-coated dishes in Glasgow minimal essential medium (Sigma), 10% knockout serum replacement, 1% modified Eagle’s medium (MEM) non-essential amino acids, 1 mM sodium pyruvate (Life Technologies), 0.1 mM 2-mercaptoethanol (Sigma), and 100 U/ml recombinant human leukemia inhibitory factor). hiPS4 and SA181 human ESC lines purchased from Cellartis and maintained in DEF-CS (Cellartis). Human neural differentiation was performed as described previously (Chambers et al., 2009 (link)), except that cells were seeded at a density of 6 × 104 cells/cm2 on Matrigel (20 μg/cm2) and grown for 48 hrs in cell medium before switching to differentiation media. Murine neural differentiation was performed as described previously (Stavridis et al., 2007 (link), Ying et al., 2003 (link)). 0.5–1.5 × 104/cm2 ESCs were plated on 0.1% gelatin-coated dishes in N2B27 (DMEM/F12; Gibco) supplemented with modified N2 (25 μg/ml insulin, 100 μg/ml apo-transferrin, 6 ng/ml progesterone, 16 μg/ml putrescine, 30 nM sodium selenite and 50 μg/ml BSA fraction V; Gibco). Medium was renewed every 2 days.
3
Maintenance of Nanog-GFP reporter ES cells
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Nanog-GFP reporter ES cells (RF8-NanogGIP #1A2 [16] ) obtained from Kyoto University were maintained on gelatin-coated dishes in the absence of feeder cells in Glasgow minimal essential medium (Sigma, St. Louis, MO, USA) supplemented with 10% FBS for ES cells (Gibco, Carlsbad, CA, USA), 1 mM sodium pyruvate, 0.1 mM 2-mercaptoethanol, nonessential amino acids (NEAA), 1000 U/mL LIF (Wako, Osaka, Japan), 50 U/mL penicillin, 50 µg/mL streptomycin, 1 µM PD0325901 (Wako), and 3 µM CHIR99021 (Cayman, Ann Arbor, MI, USA).
4
Culturing CGR8 Stem Cells for Proliferation
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The ES cell line CGR8 was cultured on gelatine-coated cell culture dishes in Glasgow Minimal Essential Medium (Sigma, Taufkirchen, Germany) to keep the cells in the undifferentiated stem cell stage for proliferation. Medium was supplemented with 9% heatinactivated (56 ° C, 30 min) fetal bovine serum (Sigma), 2 m M L -glutamine (Biochrom, Berlin, Germany), 45 μ M 2-mercaptoethanol (Sigma), and 10 3 U/mL leukemia inhibitory factor (Chemicon, Hampshire, UK) in a humidified environment containing 5% CO 2 at 37 ° C and passaged every 2-3 days. On day 0 of differentiation, adherent cells were enzymatically dissociated using 0.25% trypsin with 1 m M ethylenediaminetetra-acetic acid (EDTA) in Hank's balanced salt solution (Gibco Thermo Fisher Scientific, Darmstadt, Germany). Cells (1 × 10 7 ) were seeded in 250-mL siliconized spinner flasks (Life Technologies, Darmstadt, Germany) containing 125 mL Iscove's medium (CGR8 medium) supplemented with 16% heat-inactivated fetal bovine serum (Sigma), 100 μ M 2-mercaptoethanol (Sigma), 2 m M L -glutamine (Biochrom), and 2 m M nonessential amino acids (Biochrom). After 24 h, 125 mL medium was added to give a final volume of 250 ml. The spinner flask medium was stirred at 22.5 rpm using a stirrer system (Integra Biosciences), and 100 mL cell culture medium was exchanged every day.
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