Human Embryonic Kidney cells stably expressing the hERG channel (HEK293/hERG) were kindly provided by Dr. J. Hancox, (University of Bristol) with the permission of Dr. C.T. January, (University of Wisconsin). Cells were cultured in Dubelcco’s modified Eagle’s medium (Lonza, Basel, Switzerland), supplemented with 10% of fetal bovine serum (v/v) (Lonza, Basel, Switzerland) and 1% of penicillin-streptomycin-amphotericin cocktail (v/v) (Lonza, Basel, Switzerland) [33 (link)]. Cells were grown in the presence of 50 μmol/l of geneticin (Lonza, Basel, Switzerland). Cultures were maintained in 5% CO2 at 37 °C.
Geneticin
Manufactured by Lonza
Sourced in Spain
Geneticin is a selective antibiotic used in cell culture applications. It functions as a protein synthesis inhibitor, preventing the growth of cells that do not possess genetic resistance to the drug.
Automatically generated - may contain errors
Lab products found in correlation
Puromycin, by MP Biomedicals (2 mentions)
Geneticin, by Thermo Fisher Scientific (2 mentions)
Facsariaii, by BD (2 mentions)
Fugene hd reagent, by Promega (2 mentions)
Fetal bovine serum (fbs), by Lonza (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
β mercaptoethanol, by Merck Group (1 mentions)
L glutamine, by Lonza (1 mentions)
Pegfp puro, by Addgene (1 mentions)
5 protocols using geneticin
1
Stable hERG Expression in HEK293 Cells
2
B16-OVA Melanoma Cell Culture
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B16-OVA tumor cell line was cultured in DMEM (GIBCO®-Spain) supplemented with 10% Fetal Bovine Serum (FBS; GIBCO®-Spain), 1% penicillin/streptomycin (GIBCO®-Spain), 1% L-Glutamine (200 mM; Lonza®-Spain) and 50 μM β-mercaptoethanol (Sigma Aldrich®- Spain). This cell line, provided by Dr. D. Llopiz (CIMA, University of Navarra, Spain) was derived from B16F10 mouse syngeneic melanoma cell line transfected with chicken OVA, which its expression was maintained in presence of 400 μg/mL Geneticin (50 mg/mL, Lonza®-Spain).
3
B16OVA Melanoma Cell Line Protocol
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B16OVA murine melanoma cell line was kindly provided by Dr. Sandra Hervás-Stubbs (CIMA, Pamplona, Spain). The cell line is derived from B16-F10 and transfected with chicken ovalbumin, a protein that acts as a surrogate tumor antigen in the presence of Geneticin (50 mg/mL, Lonza®, Spain). B16OVA cells over-expressing PD-L1 [30 (link)] were maintained in a mixture of RPMI-Glutamax cell culture medium, 10% FBS, 1% P/S, 2% Hepes and 50 mM of β-mercaptoethanol under standard conditions. The presence of Mycoplasma was regularly tested (Lonza, Spain).
4
Fluorescent Cell Line Generation
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The 4T1-mScarlet and 67NR-GFP cell lines were generated by transfection using a 1:4 ratio of plasmid DNA:FugeneHD reagent (Promega), according to the manufacturer’s instructions, followed by selection with 500 µg/mL geneticin (Fisher BioReagents) and 3 µg/mL puromycin (MP Biomedicals), respectively. pmScarlet-H-C1 was a gift from Dorus Gadella (Addgene plasmid # 85043). pEGFP-puro was a gift from Michael McVoy (Addgene plasmid # 45561). The MDA-MB-231-mScarlet cell line was generated by electroporation (Lonza) of the pmScarlet-C1 plasmid, according to the manufacturer’s instructions, and selection with 500 µg/ml geneticin. After two weeks of drug selection, cells were sorted (BD FACSAriaIIµ, BD Biosciences), collecting the subpopulations expressing high levels of mScarlet or GFP.
5
Fluorescent Protein Cell Line Generation
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The 4T1-mScarlet and 67NR-GFP cell lines were generated by transfection using a 1:4 ratio of plasmid DNA:FugeneHD reagent (Promega), according to the manufacturer's instructions, followed by selection with 500 µg/mL geneticin (Fisher BioReagents) and 3 µg/mL puromycin (MP Biomedicals), respectively. pmScarlet-H-C1 was a gift from Dorus Gadella (Addgene plasmid # 85043). pEGFP-puro was a gift from Michael McVoy (Addgene plasmid # 45561). The MDA-MB-231-mScarlet cell line was generated by electroporation (Lonza) of the pmScarlet-C1 plasmid, according to the manufacturer's instructions, and selection with 500 µg/ml geneticin. After two weeks of drug selection, cells were sorted (BD FACSAriaIIµ, BD Biosciences), collecting the subpopulations expressing high levels of mScarlet or GFP.
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