All breast cancer cell lines used in this study were obtained from the Duke Cell Culture Facility (originally from ATCC). MDA-MD-361, MDA-MD-453, AU565 and BT-474 cells were cultured in RPMI1640 supplemented with 10% FBS, 2 mM glutamine, 10 mM HEPES, 1 mM sodium pyruvate, 2.5 g/L glucose and 1% penicillin-streptomycin (Thermo Fisher Scientific). UACC812 cells were cultured in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin. rBT474 cells were established as previously reported38 (link), whereas rAU565 cells were developed by 2-month selection with 1 µM lapatinib. These acquired resistant lines were maintained in 1 μM lapatinib added to regular media with supplements. All cell lines were grown at 37 °C with 5% CO2. Mycoplasma contamination was examined using Lonza MycoAlert kit (LT07). Lapatinib (L-4899) was purchased from LC Laboratories. Oxythiamine (O4000) was purchased from Sigma-Aldrich. NAC (Sigma A7250, 500 mM), fatty acids (Sigma F7050, 1:1000), or nucleosides (Millipore ES-008-D, 1:100) were incubated with the indicated cell lines for 3 days.
Es 008 d
Manufactured by Merck Group
Sourced in United States
The ES-008-D is a laboratory instrument designed for general-purpose applications. It features basic functionality for common laboratory tasks. Detailed technical specifications and intended use are not available.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Glutamax, by Thermo Fisher Scientific (2 mentions)
Esg1107, by Merck Group (2 mentions)
Penicillin streptomycin, by Merck Group (2 mentions)
Glutamax 1, by Thermo Fisher Scientific (2 mentions)
S8636, by Merck Group (2 mentions)
D6046, by Thermo Fisher Scientific (2 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Mycoalert kit, by Lonza (1 mentions)
A7250, by Merck Group (1 mentions)
Penicillin streptomycin, by Beyotime (1 mentions)
Chir99021, by Selleck Chemicals (1 mentions)
Tms001 c, by Thermo Fisher Scientific (1 mentions)
Pd0325901, by Selleck Chemicals (1 mentions)
Knockout serum replacement (ksr), by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (1 mentions)
2 mercaptoethanol, by Merck Group (1 mentions)
Mem non essential amino acid, by Corning (1 mentions)
Penicillin streptomycin, by Corning (1 mentions)
Nucleoside, by Merck Group (1 mentions)
7 protocols using es 008 d
1
Establishing Breast Cancer Cell Lines
2
Cell Culture Conditions for HEK293T, N2A, PK-15, and mESCs
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In this study, HEK293T, N2A and PK-15 cells were maintained in Dulbecco's modified Eagle medium (Yeasen, 41401ES76) supplemented with 10% fetal bovine serum (Biological Industries, 04-001-1ACS) and 1% penicillin-streptomycin (Beyotime, C0224-100ml). mESCs were cultured in Dulbecco's modified Eagle medium (Millipore, SLM-220-B) supplemented with 15% fetal bovine serum (Gibco, 10099141), 1% minimal essential medium non-essential amino acids solution (Millipore, TMS-001-C), 1% GlutaMAX (Thermo Fisher, 35050061), 1% nucleosides (Millipore, ES-008-D), 1% β-mercaptoethanol (Millipore, ES-007-E), 1 μM PD0325901 (Selleck, S1036), 3 μM CHIR99021 (Selleck, S1263) and 1,000 units ml -1 mouse leukemia inhibitory factor (Millipore, ESG1107). All cells were cultured at 37 °C in an incubator with 5% CO 2 .
3
Maintenance of Pluripotent Stem Cells
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Established cell lines were cultured and maintained in Dulbecco’s modified Eagle medium (DMEM; Sigma–Aldrich, USA, D6046) containing 10% foetal bovine serum (FBS; Gibco, USA, 16141–075), 1% penicillin-streptomycin (Sigma–Aldrich, P4333), 1% GlutaMAX-1 (Gibco, 35050–001), 1% non-essential amino acids (Gibco, 11140–050), 1% nucleosides (Millipore, USA, ES-008-D), 1% sodium pyruvate (Sigma–Aldrich, S8636), 0.1% 2-mercaptoethanol (Sigma–Aldrich), and 0.1% leukaemia inhibitory factor (LIF; Nacalai, Japan, NU0013–1) on 0.1% gelatin-coated 10-cm dishes (BD Biosciences, 353003) without feeder layers. In the +2i +LIF culture condition, the cells were cultured using DMEM with 10% knock out serum (KSR; Gibco, 10820–028) instead of 10% FBS, and supplemented with the inhibitors to MAPK and GSK3 using PD0325901 (Stemgent, USA, Stemolecule™ 04–0006) and CHIR 99021 (Stemgent, Stemolecule™ 04–0004) at 1 µM and 3 µM, respectively. Cells were passaged every 2 days.
4
Isolation and Culture of Mouse Embryonic Fibroblasts and Embryonic Stem Cells
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The MEFs were isolated from the fetuses of 14 day pregnant BALB/c mice and cultured in Dulbecco's modified Eagle's medium (DMEM; Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS; Biowest, Miami, FL, USA) and 4 mM L-glutamine. Animal experiments were carried out according to the ethics guidelines of Kyoto University. Mouse ES cells (Riken Cell Bank, Ibaragi; E14Tg2a) were cultured in DMEM (Sigma-Aldrich, USA, D6046) containing 10% FBS (Gibco, USA, 16141-075), 1% penicillin-streptomycin (Sigma-Aldrich, P4333), 1% GlutaMAX-1 (Gibco, 35050-001), 1% non-essential amino acid (Gibco, USA, 11140-050), 1% nucleosides (Millipore, USA, ES-008-D), 1% sodium pyruvate (Sigma-Aldrich, S8636), 0.1% 2-mercaptoethanol (Sigma-Aldrich), and 0.1% leukemia inhibitory factor (LIF) (Nacalai, JP, NU0013-1), on 0.1% gelatin-coated 10 cm dishes (BD Biosciences, 353003) without feeder layers.
5
Gelatin-adapted Murine Embryonic Stem Cell Culture
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For further information and requests for reagent and resources, please contact the Lead Contact, Sridhar Rao (Sridhar.rao@versiti.org; 414-937-3841).
Cell culture: Gelatin-adapted ESCs were utilized for all experiments. These are male, in-house generated, ICM-derived 129SVJ derived murine ESC line, similar to the one we have used previously and cultured under similar Serum/LIF conditions [39] (link) [40] (link). Briefly, cells were propagated under feederfree conditions in DMEM (Corning #10-017-CV) with the following supplements (FBS -GemBio #100-106, Penicillin/Streptomycin -Corning #30-002-Cl, MEM Nonessential Amino Acids -Corning #25-025-Cl, L-glutamine -Corning #25-005-Cl, Nucleosides -Sigma #ES-008-D, LIF, β-mercaptoethanol at the appropriate concentration). 2 µM of 4-Hydroxytamoxifen (4OHT) in 70% ethanol (EtOH) was used for all experiments and diluted approximately 1:1000 for drug treatments with EtOH as a control.
Cell culture: Gelatin-adapted ESCs were utilized for all experiments. These are male, in-house generated, ICM-derived 129SVJ derived murine ESC line, similar to the one we have used previously and cultured under similar Serum/LIF conditions [39] (link) [40] (link). Briefly, cells were propagated under feederfree conditions in DMEM (Corning #10-017-CV) with the following supplements (FBS -GemBio #100-106, Penicillin/Streptomycin -Corning #30-002-Cl, MEM Nonessential Amino Acids -Corning #25-025-Cl, L-glutamine -Corning #25-005-Cl, Nucleosides -Sigma #ES-008-D, LIF, β-mercaptoethanol at the appropriate concentration). 2 µM of 4-Hydroxytamoxifen (4OHT) in 70% ethanol (EtOH) was used for all experiments and diluted approximately 1:1000 for drug treatments with EtOH as a control.
6
Derivation and Maintenance of Mouse ESCs
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Mouse ESCs were cultured on mitomycin-C-treated mouse embryonic fibroblasts (MEFs) in six-well tissue culture plates (92006, TPP, Trasadingen, Switzerland). Mouse ESC maintenance medium was KnockOut™ Dulbecco’s modified Eagle medium [10829018, Thermo Fisher Scientific (Thermo)] supplemented with 20% HyClone™ fetal bovine plasma (SH30070.03, Cytiva), 1% 2-mercaptoethanol (ES-007-E, Merck), 1% nonessential amino acids (TMS-001-C, Merck), 1% nucleosides (ES-008-D, Merck), 1% L-glutamine (TMS-002-C, Merck), 100 U/mL penicillin-streptomycin (15140-122, Thermo), and 1,000 U/mL mouse leukemia inhibitory factor (ESG1107, Merck). Before use, 1 μM mirdametinib [PD0325901/162-25291, FUJIFILM Wako (Wako)] and 3 μM laduviglusib (CHIR-99021/TB4423-GMP, Bio-Techne) were added to mESC maintenance medium. Cells were passaged every 2 to 3 d.
For cryopreservation, CELLBANKER 1plus (CB023, Takara) was used. For thawing, 1 mL of phosphate-buffered saline (PBS, Wako, 048-29805) was added to a cryopreserved cell vial. Mouse ESCs were quickly dispersed by pipetting and transferred to 15-mL centrifuge tubes containing 9 mL of PBS. After centrifugation, the cells were suspended in mESC maintenance medium containing 1 μM PD0325901 and 3 μM CHIR-99021. The cell suspension was then seeded onto MEF feeder cells. The thawed mESCs were used for injection after more than one passage.
For cryopreservation, CELLBANKER 1plus (CB023, Takara) was used. For thawing, 1 mL of phosphate-buffered saline (PBS, Wako, 048-29805) was added to a cryopreserved cell vial. Mouse ESCs were quickly dispersed by pipetting and transferred to 15-mL centrifuge tubes containing 9 mL of PBS. After centrifugation, the cells were suspended in mESC maintenance medium containing 1 μM PD0325901 and 3 μM CHIR-99021. The cell suspension was then seeded onto MEF feeder cells. The thawed mESCs were used for injection after more than one passage.
7
Culturing B6xCAST Mouse Embryonic Stem Cells
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mESC were cultured as previously described (Brosh et al. 2023 (link)). Specifically, C57BL/6J × CAST/EiJ (B6xCAST) mESCs were cultured on plates coated with 0.1% gelatin (EMD Millipore ES006-B) in 80/20 medium comprising 80% 2i medium and 20% mESC medium. 2i medium contained a 1:1 mixture of Advanced DMEM/F12 (ThermoFisher 12634010) and Neurobasal-A (ThermoFisher 10888022) supplemented with 1% N2 Supplement (ThermoFisher 17502048), 2% B27 Supplement (ThermoFisher 17504044), 1% GlutaMAX (ThermoFisher 35050061), 1% Pen-Strep (ThermoFisher 15140122), 0.1 mM 2-mercaptoethanol (Sigma M3148), 1,250 U/mL LIF (ESGRO ESG1107l), 3 μM CHIR99021 (R&D Systems 4423), and 1 μM PD0325901 (Sigma PZ0162). mESC medium contained knockout DMEM (ThermoFisher 10829018) supplemented with 15% FBS (BenchMark 100–106), 0.1 mM 2-mercaptoethanol, 1% GlutaMAX, 1% MEM nonessential amino acids (ThermoFisher 11140050), 1% nucleosides (EMD Millipore ES008-D), 1% PenStrep, and 1,250 U/mL LIF. Cells were grown at 37 °C in a humidified atmosphere of 5% CO2 and passaged twice per week on average.
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