DHFR sufficient CHO wild type adherent cell line (CHO-WT) was a generous gift from Dr. Gunes Ozhan (Izmir Biomedicine and Genome Center). These cells were grown in DMEM/F-12 medium (11320-033, Gibco) supplemented with 10% FBS (10500064, Gibco) and 1% Penicillin/Streptomycin (15140122, Gibco). DHFR deficient CHO-DG44 adherent cells, obtained as a kind gift from Dr. Lawrence Chasin (Columbia University), were cultured in Alpha MEM Eagle medium (BE12-169F, Lonza) supplemented with 10% FBS and 1% Penicillin/Streptomycin. Both cell lines were maintained at 37 °C and 5% CO2 cell culture conditions. CHO-DG44 suspension cells, adapted in-house from CHO-DG44 adherent cells to grow in serum-free suspension culture, were cultivated in CD DG44 medium (12610-010, Gibco) supplemented with L-glutamine at 8 mM final concentration and 18 ml/L (1.8%) Pluronic F-68 Non-ionic Surfactant (24040032, Gibco), and maintained at 37 °C and 8% CO2 cell culture conditions and passaged every 3–4 days by dilution. Cells were routinely tested negative for mycoplasma contamination using a PCR-based mycoplasma detection kit (G238, ABM).
Cd dg44 medium
Manufactured by Thermo Fisher Scientific
Sourced in United States
CD DG44 medium is a chemically defined, protein-free cell culture medium formulated for the growth and maintenance of Chinese Hamster Ovary (CHO) DG44 cells. It is designed to support cell proliferation and recombinant protein production in suspension culture.
Automatically generated - may contain errors
Lab products found in correlation
L glutamine, by Thermo Fisher Scientific (3 mentions)
Pluronic f 68, by Thermo Fisher Scientific (3 mentions)
Freestyle max reagent, by Thermo Fisher Scientific (2 mentions)
Cho dg44, by Thermo Fisher Scientific (2 mentions)
Cd opticho, by Thermo Fisher Scientific (2 mentions)
Pluronic f 68 non ionic surfactant, by Thermo Fisher Scientific (1 mentions)
Dmem f12 medium, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Lonza (1 mentions)
Dmem f12, by Thermo Fisher Scientific (1 mentions)
Amaxa nucleofector 2 device, by Lonza (1 mentions)
Amaxa cell line nucleofector kit 5, by Lonza (1 mentions)
5 protocols using cd dg44 medium
1
Culturing CHO Cell Lines for Protein Production
2
Culturing Adherent and Suspension CHO Cells
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CHO-K1 cells were cultured in adherent conditions in the chemically defined medium DMEM/F12 (Gibco, USA), supplemented with 5% (v/v) of fetal bovine serum (FBS) (HyClone, USA) and 100 mM of L-glutamine (Gibco, USA). Suspension-adapted, dihydrofolate reductase (DHFR)-deficient CHO DG44 cells were obtained from ThermoFisher Scientific (USA) and initially cultured in a chemically defined CD-DG44 medium (Gibco, USA) with a mixture of sodium hypoxanthine and thymidine (HT).
3
Suspension CHO Cell Transfection and Amplification
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Suspension CHO (CHO DG44, Gibco, hereinafter termed susCHO) cells were cultured in CD DG44 medium (Gibco) supplemented with 8 mM l -glutamine (Gibco) and 0.18% Pluronic® F-68 prior to transfection. Transfection was performed by combining 18 μg AhdI-linearized plasmid pOpti_IL2_S377-588-Fc and 15 μL of FreeStyle™ MAX Reagent (Invitrogen) in 1.2 mL OptiPRO SFM and incubated at room temperature for 10 min, followed by dropwise addition to 1.5 × 107 cells in 30 mL of CD DG44 culture medium (non-selective) according to the manufacturer’s instructions. After 48 h, cells were transferred to selective medium (CD OptiCHO, Invitrogen), supplemented with 8 mM l -glutamine and 0.18% Pluronic® F-68 (Gibco), and cultivated until cell viability reached 90%. After selection, stably transfected susCHO cells underwent DNA amplification by gradually increasing MTX concentration (20–5000 nM) in selective medium. All suspension culture flasks were maintained in a humidified incubator, 37 °C/8% CO2 on a shaker, at a constant rotation rate of 135 rpm.
4
Suspension CHO Cell Transfection and Amplification
5
Efficient CHO Cell Transfection Protocol
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CHO DG44 host cells were cultured in CD-DG44 medium (Gibco, Thermo Fisher Scientific) supplemented with 6 mM L-Glutamine and Pluronic F-68 (Gibco, Thermo Fisher Scientific). For electroporation, the Amaxa Cell Line Nucleofector Kit V and Amaxa Nucleofector II device were used (Lonza Group) according to the manufacturer's protocol. Briefly, cells were electroporated with linearized plasmid DNA.
For each construct a total of 10 independent transfection reactions were carried out and then pooled together into T-175 flasks. After 24 h the pooled culture was split into T-25 flasks and transferred into selective medium. The resulting cell pools were then adapted back into shaking culture.
For each construct a total of 10 independent transfection reactions were carried out and then pooled together into T-175 flasks. After 24 h the pooled culture was split into T-25 flasks and transferred into selective medium. The resulting cell pools were then adapted back into shaking culture.
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