HEK293T and HEK293 cell lines were maintained in high glucose Dulbecco’s Modified Eagle Medium, supplemented with 10% fetal bovine serum (FBS), 0.1 mM MEM nonessential amino acids (NEAA), 2 mM l -glutamine, and 1% penicillin/streptomycin. The E14 cell line of mESCs was cultured on precoated 0.2% gelatin (Sigma) tissue culture dishes in Glasgow Minimum Essential Medium, also containing 15% FBS (Biowest), Tylosine (Sigma), 0.1 mM β-mercaptoethanol (Gibco), supplemented with nonessential amino acids (Gibco), 1 mM sodium pyruvate (Gibco), LIF conditioned media, and GlutaMAX (Gibco). LIF-conditioned media was prepared as previously described36 (link). All cell lines were grown at 37 °C in a 5% CO2 incubator and passaged every 2–3 days. For mESC differentiation, the cells were first washed with phosphate-buffered saline, then incubated in media without LIF, and harvested at the indicated time of differentiation. For EB differentiation, EBs were generated through trypsinization of mESC cultures and plating 1000 cells/drop using the hanging drop technique on a low attachment petri dish (Corning) in ESC medium lacking LIF. EBs were then transferred to a tissue culture dish after three days for further differentiation and harvested at the indicated time point.
Tylosine
Manufactured by Merck Group
Sourced in Germany
Tylosine is a macrolide antibiotic produced by the bacterium Streptomyces fradiae. It is used as a veterinary drug for the treatment of bacterial infections in livestock and poultry.
Automatically generated - may contain errors
Lab products found in correlation
Sodium pyruvate, by Thermo Fisher Scientific (2 mentions)
Glutamax, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Biowest (2 mentions)
β mercaptoethanol, by Thermo Fisher Scientific (2 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (1 mentions)
Low attachment petri dish, by Corning (1 mentions)
Nonessential amino acid, by Corning (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Tissue culture plates, by Merck Group (1 mentions)
Mia paca 2 cell, by American Type Culture Collection (1 mentions)
Rpmi 1640 medium, by Merck Group (1 mentions)
Amphotericin b, by Harvard Bioscience (1 mentions)
3 protocols using tylosine
1
Maintenance and Differentiation of Mammalian Cell Lines
2
Mouse and Human Cell Culture Protocols
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Mouse E14 embryonic stem cells (mESCs) and human embryonic kidney HEK293T cells were used for the experiments. mESCs were cultured on tissue culture plates (Sigma) pre-coated with 0.2% gelatin at 37°C in a 5% CO2 incubator. Cell culture media are consisted of 15% fetal bovine serum (FBS, Biowest), 1 mM sodium pyruvate (GIBCO), 100X non-essential amino acid (Corning), 0.1 mM β-mercaptoethanol (GIBCO), 100X GlutaMAX (GIBCO), Tylosine (Sigma), and 1,000 U/mL of LIF (conditioned media). LIF-containing media were produced from Cos7 cells transfected with LIF cDNA by Lipofectamine. Subculture was performed by 0.01% trypsin treatment for 3 min in a 37°C incubator. After incubation, trypsin was neutralized by FBS-containing media, and mESCs were collected by centrifugation. Subsequently, mESCs were plated on a pre-coated culture dish. HEK293T cells were maintained on a SPL tissue culture plate with Dulbecco’s Modified Eagle’s Medium (DMEM). Media contained 10% fetal bovine serum (FBS, Gibco) and 1X antibiotics (anti-anti, Gibco). Subculture was performed with 0.05% trypsin. Cells were re-suspended with FBS-containing media for neutralization and re-plated on a tissue culture plate. Calcium phosphate method was used for plasmid DNA transfection.
3
Culture of Pancreatic Cancer Cell Lines
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The 6606PDA cells, containing a Kras G12D mutation (a gift from Prof. Tuveson, University of Cambridge, UK) [23 (link)], were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, 4.5 g/l Glucose, Biochrom GmbH, Berlin, Germany), supplemented with 10% fetal calf serum (FCS), 1 ml/l Tylosine (8 mg/ml, Sigma-Aldrich) and 1 ml/l Amphotericin B (250 μg/ml, Biochrom GmbH). The national cancer institute provided the murine Panc02 cells. The human Mia Paca-2 cells were purchased from ATCC (Manassas, VA, USA). These two cell lines were cultured in RPMI 1640 medium (Sigma-Aldrich), supplemented with 10% FCS, penicillin and streptomycin.
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