Human hTert immortalized NER proficient VH10 fibroblasts, GG-NER deficient XP-C-fibroblasts XP186LV, XP3MA and XP20MA, NER deficient XP-A (XP25RO) and TC-NER deficient CS-B fibroblasts (CS1AN) were cultured in Ham's F10 medium (Lonza) supplemented with 15% fetal calf serum (Biowest) and 1% penicillin–streptomycin (Sigma-Aldrich) at 37°C with 5% CO2 in a humidified incubator. All NER deficient cells were characterized either by complementation studies or by mutation analysis (32 (link),33 (link)). For transcription inhibition, cells were pretreated either with 25 μg/ml α-amanitin (Sigma-Aldrich) for 16 h or 100 μM 5,6-dichloro-1-β-d -ribofuranosylbenzimidazole (DRB, Calbiochem) for 1 h before DNA damage infliction. For inhibiting DNA polymerase ß, pamoic acid (PA, Sigma-Aldrich) dissolved in 200 mM NaCl and 20 mM Tris–HCl (pH 7) was added to the cells in a final concentration of 500 μM 16 h before the experiment was started. Fresh PA was added during EdU labeling.
Ham s f10 medium
Manufactured by Lonza
Ham's F10 medium is a cell culture medium formulated to support the growth and maintenance of a variety of cell types. It provides a balanced combination of nutrients, salts, and supplements required for cell proliferation. The medium is commonly used in a range of cell biology applications, but a detailed description of its specific intended use is not available.
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Lab products found in correlation
α amanitin, by Merck Group (1 mentions)
Penicillin streptomycin, by Merck Group (1 mentions)
Fetal calf serum (fcs), by Biowest (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Euroclone (1 mentions)
Dmem high glucose with l glutamine, by Euroclone (1 mentions)
Fungizone, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Hepes buffer, by Lonza (1 mentions)
Dmem with l glutamine, by Lonza (1 mentions)
Amaxa nhdf nucleofector kit, by Lonza (1 mentions)
Fetal bovine serum (fbs), by Lonza (1 mentions)
3 protocols using ham s f10 medium
1
NER-Deficient Fibroblast Cell Culture
2
Fibroblast Culture Protocol for NPS-TTD
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Individuals investigated in this study followed the protocols approved by institute-specific ethical review boards, and informed consents were obtained for biological samples according to the Helsinki guidelines.
Primary skin fibroblasts from the NPS-TTD cases TTD236AM, TTD1GL, TTD17PV, TTD18PV, from XP25RO (XP-A) and from the genetically unrelated healthy donors C3PV, C1609RM, C5BO, C4RO and C5RO were used. Fibroblasts were cultured in DMEM medium (DMEM High Glucose with L-glutamine, EuroClone) or Ham’s F10 medium (Lonza) supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin–streptomycin (EuroClone) at 37°C and 5% CO2.
Primary skin fibroblasts from the NPS-TTD cases TTD236AM, TTD1GL, TTD17PV, TTD18PV, from XP25RO (XP-A) and from the genetically unrelated healthy donors C3PV, C1609RM, C5BO, C4RO and C5RO were used. Fibroblasts were cultured in DMEM medium (DMEM High Glucose with L-glutamine, EuroClone) or Ham’s F10 medium (Lonza) supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin–streptomycin (EuroClone) at 37°C and 5% CO2.
3
Functional Complementation of ACBD5 in Fibroblasts
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Primary skin fibroblasts and HeLa cells were cultured in Ham's F-10 medium (Lonza, Basel, Switzerland) or Dulbecco's modified Eagle's medium (DMEM) with L-glutamine (Bio-Whittaker) supplemented with 10% fetal bovine serum (Bio-Whittaker), 25 mM HEPES buffer (BioWhittaker), 100 U/ml penicillin, 100 mg/ml streptomycin (Life Technologies), and 250 ng/ml fungizone (Life Technologies) in a humidified atmosphere of 5% CO 2 at 37⁰C. Transfection of HeLa cells was performed in 6-well plates using the jetPRIME® DNA transfection kit (Polyplus transfection, Illkirch-Graffenstaden, France) according to the manufacturer's instructions. Transfection of skin fibroblasts was performed using the AMAXA NHDF Nucleofector Kit (Lonza, Basel, Switzerland) according to the manufacturer's instructions (program U23).
For functional complementation we transfected patient's skin fibroblasts with the plasmid pcDNA3.1-hsACBD5, containing full-length human ACBD5 cDNA (1.5kb). The medium was changed 24 hours after transfection. For immunofluorescence microscopy, cells were imaged 3 days after transfection. For the D3-C22:0 loading test, D3-C22:0 was added to the medium one day after transfection and fatty acid analysis was performed three days later. C26:0 lysoPC was measured in cell pellets four days after transfection.
For functional complementation we transfected patient's skin fibroblasts with the plasmid pcDNA3.1-hsACBD5, containing full-length human ACBD5 cDNA (1.5kb). The medium was changed 24 hours after transfection. For immunofluorescence microscopy, cells were imaged 3 days after transfection. For the D3-C22:0 loading test, D3-C22:0 was added to the medium one day after transfection and fatty acid analysis was performed three days later. C26:0 lysoPC was measured in cell pellets four days after transfection.
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