Collagen type 1 (rat tail) was from Millipore or from PureCol® (bovine) (Advanced Biomatrix, USA). The CPT1a inhibitor (+)-etomoxir sodium salt hydrate was purchased from CNIO Carlos III Therapies. Mitomycin C (MitoC), sodium palmitate, dimethyl sulfoxide (DMSO), NAC, sodium acetate, oligomycin, cycloheximide, cytidine, adenine, guanosine, methotrexate, carnitine, dNTP mix and tamoxifen were from Sigma-Aldrich (Bornem, Belgium). 5-fluorouracil (TEVA Pharma Belgium) was obtained from the pharmacy of the university hospital Leuven. Nucleoside mix was from Millipore (Belgium)15 . Hoechst 33342 and L-homopropargylglycine (HPG) were from Molecular Probes and L-glutamine and penicillin/streptomycin were from Gibco® (Invitrogen, Life Technologies, Ghent, Belgium). Uniformly labeled [U-13C]-potassium palmitate, [U-13C]-acetate, [U-13C]-glucose, [U-13C]-glutamine and [U-13C]-algal fatty acid mix were obtained from Cambridge isotope laboratories, Inc. [U-14C]-palmitate, [9,10-3H]-palmitate, [6-14C]-D-glucose, [8-14C]-hypoxanthine and [3H]-thymidine were from Perkin Elmer.
Nucleoside mix
Manufactured by Merck Group
Sourced in Belgium
Nucleoside mix is a laboratory reagent containing a combination of nucleosides, which are the basic structural units of nucleic acids such as DNA and RNA. This product is used in various applications within the field of molecular biology and biochemistry, where the study and manipulation of nucleic acids are essential.
Automatically generated - may contain errors
Lab products found in correlation
Glutamax, by Thermo Fisher Scientific (6 mentions)
L glutamine, by Thermo Fisher Scientific (6 mentions)
Penicillin streptomycin solution, by Thermo Fisher Scientific (4 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (4 mentions)
Recombinant lif, by Merck Group (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (3 mentions)
Cytidine, by Merck Group (2 mentions)
Guanosine, by Merck Group (2 mentions)
Carnitine, by Merck Group (2 mentions)
Methotrexate, by Merck Group (2 mentions)
Oligomycin, by Merck Group (2 mentions)
Dntp mix, by Merck Group (2 mentions)
U 13c glucose, by Cambridge Isotopes (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
U 13c glutamine, by Cambridge Isotopes (2 mentions)
Purecol, by Advanced BioMatrix (2 mentions)
U 13c acetate, by Cambridge Isotopes (2 mentions)
L homopropargylglycine hpg, by Thermo Fisher Scientific (2 mentions)
Uniformly labeled u 13c potassium palmitate, by Cambridge Isotopes (2 mentions)
15 protocols using nucleoside mix
1
Metabolic Labeling Techniques in Cell Culture
2
Culture of Engineered Stem Cells
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ESCs expressing 3 × FLAG and biotin tagged proteins were maintained in complete ESC culture medium: DMEM (Dulbecco's modified Eagle's medium) supplemented with 15% heat-inactivated FCS (fetal calf serum), 2 mM GlutaMax (100 × Life Technology), 1% Nucleoside mix (100 × stock, Millipore), penicillin–streptomycin solution (100 × stock, Life Technologies), 0.1 mM non-essential amino acids (NEAA), 0.1 mM β-mercaptoethanol and supplied with 1000 U/ml recombinant Leukemia Inhibitory Factor (LIF, Millipore). ESCs were cultured on plates which were pre-coated with 0.1% gelatin.
3
Metabolic Labeling Techniques in Cell Culture
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Collagen type 1 (rat tail) was from Millipore or from PureCol® (bovine) (Advanced Biomatrix, USA). The CPT1a inhibitor (+)-etomoxir sodium salt hydrate was purchased from CNIO Carlos III Therapies. Mitomycin C (MitoC), sodium palmitate, dimethyl sulfoxide (DMSO), NAC, sodium acetate, oligomycin, cycloheximide, cytidine, adenine, guanosine, methotrexate, carnitine, dNTP mix and tamoxifen were from Sigma-Aldrich (Bornem, Belgium). 5-fluorouracil (TEVA Pharma Belgium) was obtained from the pharmacy of the university hospital Leuven. Nucleoside mix was from Millipore (Belgium)15 . Hoechst 33342 and L-homopropargylglycine (HPG) were from Molecular Probes and L-glutamine and penicillin/streptomycin were from Gibco® (Invitrogen, Life Technologies, Ghent, Belgium). Uniformly labeled [U-13C]-potassium palmitate, [U-13C]-acetate, [U-13C]-glucose, [U-13C]-glutamine and [U-13C]-algal fatty acid mix were obtained from Cambridge isotope laboratories, Inc. [U-14C]-palmitate, [9,10-3H]-palmitate, [6-14C]-D-glucose, [8-14C]-hypoxanthine and [3H]-thymidine were from Perkin Elmer.
4
Maintenance of Embryonic Stem Cells
5
Culturing and Differentiating Embryonic Stem Cells
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WT (46C) and various KO and KI ESCs were cultured on gelatin-coated plates in standard ESC culture medium: Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 15% heat-inactivated fetal bovine serum (Hyclone), 1% penicillin–streptomycin (Cellgro), 1% of nucleoside mix (100× stock, Millipore), 1% Glutamax (GIBCO), 1% MEM nonessential amino acids (Cellgro), 0.1 mM 2-mercaptoethanol, and 1000 U/ml recombinant LIF (Millipore). For differentiation time course with LIF withdrawal, cells were harvested at Day 0, Day 2, Day 4, and Day 6 of differentiation.
6
Maintenance of Embryonic Stem Cells
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ESCs (CJ9 and 46C) were maintained in DMEM (Dulbecco’s modified Eagle’s medium) supplemented with 15% heat-inactivated FCS (fetal calf serum), 2 mM Glutamax (100 × Life Technology), 1% nucleoside mix (100 × stock, Millipore), 1 × Penicillin-Streptomycin Solution (100 × stock, Life Technologies), 0.1 mM non-essential amino acids, 0.1 mM 2-mercaptoethanol and supplied with 1000 U/ml recombinant leukemia inhibitory factor (LIF, Millipore). HEK293T cells and MEF cells were cultured in DMEM medium containing 10% FCS and 1 × Penicillin-Streptomycin Solution.
7
Mouse J1 ES Cell Culture Maintenance
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Mouse J1 ES cell lines were maintained as described previously24 (link). In detail, cells were maintained in ES medium (Dulbecco’s modified Eagle’s medium) supplemented with 15% fetal calf serum, 0.1 mM β-mercaptoethanol, 2 mM L -glutamine, 0.1 mM non-essential amino acid, 1% of nucleoside mix (100 × stock, Millipore), 1,000 U ml−1 recombinant leukaemia inhibitory factor (Chemicon) and 50 U ml−1 penicillin/streptomycin).
8
Detailed Protocols for Murine Embryonic Stem Cell Culture
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ESCs (CJ9, 46C and cells expressing 3X FLAG and biotin tagged RBPs) were maintained in complete ESC culture medium: DMEM (Dulbecco's modified Eagle's medium) supplemented with 15% heat-inactivated FCS (fetal calf serum), 2 mM Glutamax (100 3 Life Technology), 1% nucleoside mix (100 3 stock, Millipore), Penicillin-Streptomycin Solution (100 3 stock, Life Technologies), 0.1 mM non-essential amino acids, 0.1 mM 2-mercaptoethanol and supplied with 1000 U/mL recombinant leukemia inhibitory factor (LIF, Millipore). ESCs were cultured on plates which were pre-coated with 0.1% gelatin. ESCs used in this study are male. The HEK293T cells and MEF cells were cultured in medium containing DMEM, 10% FCS, 1 3 Penicillin-Streptomycin Solution.
All mice experiments were conducted in accordance with the Guide for the Care and Use of Animals for Research Purposes in Tsinghua University. Zygotes were collected from super ovulated C57BL/6 females mated with C57BL/6 males. Zygotes were collected in M2 medium (Sigma) and cultured in KSOM medium (Millipore).
All mice experiments were conducted in accordance with the Guide for the Care and Use of Animals for Research Purposes in Tsinghua University. Zygotes were collected from super ovulated C57BL/6 females mated with C57BL/6 males. Zygotes were collected in M2 medium (Sigma) and cultured in KSOM medium (Millipore).
9
Culturing Mouse Embryonic Stem Cells
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Mouse embryonic stem cells (mESCs) J1 (strain 129S4/SvJae, RRID:CVCL_6412 ,) and CJ7 (strain 129S1/SvImJ, RRID:CVCL_C316 ) were cultured on 0.1% gelatin-coated plates in ESM medium: DMEM supplemented with 15% fetal bovine serum (FBS), 1000 units/mL recombinant leukemia inhibitory factor (LIF), 0.1 mM 2-mercaptoethanol, 2 mM L-glutamine, 0.1 mM MEM non-essential amino acids (NEAA), 1% nucleoside mix (100X stock, Sigma), and 50 U/mL Penicillin/Streptomycin). Primed mouse epiblast stem cells (EpiSCs) were culture on fibronectin-coated plates (10 μg/mL/cm2) in N2B27 medium supplemented with Activin A (20 ng/mL) and Fgf2 (12 ng/mL) (Fidalgo et al., 2016 (link)). Generation of Zfp281KO ESCs (from CJ7 background, clone no. 2.6 (XX), 3.34 (XY) and 7 (XX)) has been previously described (Fidalgo et al., 2011 (link)). All cell lines are from authenticated sources and mycoplasma contamination test was performed routinely.
10
Culturing Mouse Embryonic Stem and Trophoblast Stem Cells
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Mouse J1 ES cells were cultured in ES+ media, composed of DMEM (Dulbecco's modified Eagle's medium) supplemented with 18% fetal bovine serum (FBS), 2mM L-glutamine, 100 μM of non-essential amino acid supplement, nucleoside mix (100× stock, Sigma), 100 μM of β-mercaptoethanol, 1000U/ml of recombinant leukemia inhibitory factor (LIF, Chemicon) and 50U/ml of penicillin/streptomycin. ES cells were plated on 0.1% gelatin coated dishes. Mouse TS cells were maintained in TS+ media, at a ratio of 3:7 of TS medium to mouse embryonic fibroblasts (MEF)-conditioned TS medium, supplemented with 25 ng/ml Fgf4 and 1 μg/ml heparin. TS medium is RPMI 1640 (Roswell Park Memorial Institute medium, Gibco) supplemented with 20% FBS, 100 μM β-mercaptoethanol, 2 mM L-glutamine, 1 mM sodium pyruvate, penicillin (50U/ml) and streptomycin (50 mg/ml). MEF-conditioned medium is TS medium conditioned by MEF. MEF were treated with mitomycin, followed by culturing for 3 days. The medium was collected every 3 days for three times. 293T cells were maintained in DMEM supplemented with 10% FBS, 2 mM L-glutamine and 50U/ml of penicillin/streptomycin. All cells were incubated at 37°C, 5% CO2.
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