Niacin and tryptophan free RPMI1640 media from Corning Life Sciences (Tewksbury, MA), was reconstituted with natural isotopic abundance 2 mg L−1 niacin, 5 mg L−1 tryptophan, 2 mg L−1 [13C11]-tryptophan and/or 5 mg L−1 [13C315N1]-nicotinamide as indicated for each experimental group. HepG2 human hepatocellular carcinoma cells were obtained from ATCC, and passaged at 80% confluence. Fetal bovine sera (FBS) that were dialyzed (dFBS) or charcoal-stripped (csFBS) were purchased from Golden West Biological (Temecula, CA). Reduced amounts of serum in the media increased doubling time in culture after the first passage. The protocol was therefore modified to passage cells in media containing 10% serum, then to perform an overnight incubation in 2% serum containing media before harvesting the cells for use as an internal standard. This “ultra-labeling” has been used successfully before in pantothenate SILEC.12 Trp5 mutant S. cerevisiae (Carolina Biological Supply, Burlington, NC) was cultured in standard defined medium as previously described13 (link) but omitting tryptophan and niacin, which was then replaced with labeled analogs as above. Yeast culture was grown to the stationary phase, then harvested for extraction.
Hepg2 human hepatocellular carcinoma cells
Manufactured by American Type Culture Collection
Sourced in United States
The HepG2 human hepatocellular carcinoma cells are a well-established cell line derived from a human liver cancer. They are commonly used in various research applications, including the study of liver function, metabolism, and drug development. The HepG2 cells provide a reliable and reproducible model for in vitro investigations.
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Lab products found in correlation
Penicillin, by Thermo Fisher Scientific (3 mentions)
Streptomycin, by Thermo Fisher Scientific (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Rpmi 1640 media, by Corning (1 mentions)
Penicillin, by American Type Culture Collection (1 mentions)
Streptomycin, by American Type Culture Collection (1 mentions)
Ea hy926 human endothelial cells, by American Type Culture Collection (1 mentions)
Dulbecco modified eagle medium (dmem), by Harvard Bioscience (1 mentions)
Hydroxytamoxifen, by Merck Group (1 mentions)
Methotrexate, by Bio-Techne (1 mentions)
Cell culture plates, by Thermo Fisher Scientific (1 mentions)
Dimethyl sulfoxide (dmso), by Thermo Fisher Scientific (1 mentions)
Mda mb 231 human breast cancer cells, by American Type Culture Collection (1 mentions)
Celltiter blue ctb, by Promega (1 mentions)
Cell culture medium, by Thermo Fisher Scientific (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Thermo Fisher Scientific (1 mentions)
Calcein am, by Thermo Fisher Scientific (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
3t3 l1 mouse embryo fibroblasts, by American Type Culture Collection (1 mentions)
Microplate reader, by Agilent Technologies (1 mentions)
11 protocols using hepg2 human hepatocellular carcinoma cells
1
Isotopic Labeling of Niacin and Tryptophan
2
Immortalized Murine Microvascular Endothelial Cells
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b.End3 murine microvascular endothelial cells were obtained from the European Collection of Cell Cultures (ECACC, Salisbury, UK). The b.End3 cells were established form brain endothelial cells of 129/Sv mice by immortalization with the Polyoma virus middle T-antigen [29 (link)] . The cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Biochrom AG, Berlin, Germany) containing 1g/l glucose supplemented with 10% fetal bovine serum (FBS, Hyclone, Logan, UT), 1% non-essential amino acids, 100 IU/ml penicillin and 100 μg/ml streptomycin (Invitrogen, Carlsbad, CA) at 37°C in 10% CO2 atmosphere [28 (link)] . bEnd.3 microvascular cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA) and maintained similarly. This cell line was established by infecting BALB/c cortical capillary endothelial cells with the Polyoma middle T-antigen expressing retrovirus [30 (link), 31 (link)] . EA.hy926 human endothelial cells were obtained from ATCC (Manassas, VA) and maintained in DMEM containing 1 g/l glucose supplemented with 10% FBS, 1% non-essential amino acids, 100 IU/ml penicillin and 100 μg/ml streptomycin. HepG2 human hepatocellular carcinoma cells were obtained from ATCC (Manassas, VA) and maintained in Eagle’s minimum essential medium (EMEM, Cellgro Mediatech Inc., Manassas, VA) supplemented with 10% FBS, 100 IU/ml penicillin and 100 μg/ml streptomycin.
3
Diverse Cell Lines and Reagents for Biomedical Research
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We purchased Jurkat human T-lymphoblast cells, EL4 mouse T-lymphoblast cells, HepG2 human hepatocellular carcinoma cells, MDA-MB231 human breast cancer cells, and Ln229 human glioblastoma cells from ATCC (Manassas, VA). We also purchased the following: Bodipy®.FL.L-cystine (BFC) from Life technologies (Grand Island, NY); Propidium Iodide (PI) from Sigma-Aldrich (Milwaukee, WI); Alexa Fluor Annexin V from BioLegend (San Diego, CA); Cell Titer Blue (CTB) from Promega Corporation (Madison, WI); MTT from Life Technologies (Eugene, OR); DCFDA from Sigma-Aldrich (Milwaukee, WI); Calcein AM from Life Technologies (Eugene, OR); etoposide, paclitaxel and hydroxyTamoxifen from Sigma-Aldrich (St. Louis, MO); methotrexate from Tocris Bioscience (Bristol, UK); DMSO from Fisher Scientific (Santa Clara, CA); and cell culture medium, FBS, penicillin, streptomycin, sodium bicarbonate, and all cell culture plates from GIBCO BRL (Frederick, MD). Plasmid vector expressing p53 protein under a Tamoxifen regulatable gene expression system was constructed by standard gene cloning procedures in our laboratory29 (link).
4
Culturing HepG2 Hepatocellular Carcinoma Cells
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The HepG2 human hepatocellular carcinoma cells were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were cultured in Dulbecco modified eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), penicillin (100 IU/mL), and streptomycin (100 μg/mL) (Gibco, ThermoFisher Scientific Co., Waltham, MA, USA). The cells were incubated in an incubator containing a humidified atmosphere of 5% CO2 at 37 °C.
5
Meratrim Treatment on Adipocytes and Hepatocytes
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3T3-L1 mouse embryo fibroblasts and HepG2 human hepatocellular carcinoma cells were obtained from American Type Culture Collection (Manassas, VA) and cultivated in DMEM supplemented with 10 % fetal bovine serum (FBS) 100 U/ml penicillin, 100 μg/ml streptomycin, 1 mM sodium pyruvate and 4.5 g/L D-glucose. 3T3-L1 preadipocytes were differentiated to mature adipocytes as described previously [8 (link)]. For treatments, the dry powdered Meratrim was dissolved in DMSO and the final concentration of DMSO in the culture was 0.2 % (v/v) in all experiments. Matured adipocytes or hepatocytes were treated with desired concentration of Meratrim for various time periods; vehicle control culture wells received 0.2 % DMSO only.
6
Protective Effect of Recombinant PPI Protein on HepG2 Cells
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HepG2 human hepatocellular carcinoma cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were cultured at 37°C in a humidified 5% CO2, 95% air equilibrated incubator in minimum essential medium (MEM) supplemented with heat-inactivated 10% fetal bovine serum (HyClone, Logan, UT, USA), penicillin (100 U-ml-1) and streptomycin (100 µg-ml-1). Adherent cells at 70–80% confluence were detached by trypsin-EDTA solution and re-plated.
Cell viability was estimated with the CytoX Cell Viability Assay kit (LPS Solution, Daejeon, Korea). Briefly, cells (1.0×103 cells/well) were seeded in a 96-well plate. Various concentrations (0.001, 0.01, 0.1 and 1 µg-ml−1) of recombinant PPI protein were added to the cells along with 1 mM H2O2 for 1 h at 37°C. Following incubation, Cyto solution was added to each well and incubated for an additional 1 h at 37°C. Absorbance was measured with a microplate reader (BioTek Instruments, Inc., Winooski, VT, USA) at a wavelength of 450 nm. The viability of purified recombinant PPI protein-treated cells was expressed as a percentage of that in negative control cells (−, without H2O2).
Cell viability was estimated with the CytoX Cell Viability Assay kit (LPS Solution, Daejeon, Korea). Briefly, cells (1.0×103 cells/well) were seeded in a 96-well plate. Various concentrations (0.001, 0.01, 0.1 and 1 µg-ml−1) of recombinant PPI protein were added to the cells along with 1 mM H2O2 for 1 h at 37°C. Following incubation, Cyto solution was added to each well and incubated for an additional 1 h at 37°C. Absorbance was measured with a microplate reader (BioTek Instruments, Inc., Winooski, VT, USA) at a wavelength of 450 nm. The viability of purified recombinant PPI protein-treated cells was expressed as a percentage of that in negative control cells (−, without H2O2).
7
Establishing Cell Lines for Cancer Research
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HepG-2 human hepatocellular carcinoma cells were purchased from American Type Culture Collection (ATCC) and transfected with luciferase-GFP lentivirus, and the selected pure GFP-labeled cells were sorted with a Moflo-XDP high-speed flow cell sorter and cultured with Eagle's minimum essential medium (EMEM) supplemented with 10% fetal bovine serum. Human NCI-H520 and NCI-H446 cells were purchased from ATCC and cultured with Roswell Park Memorial Institute (RPMI)-1640 supplemented with 10% fetal bovine serum.
8
HepG2 Cell Culture and Oleic Acid Treatment
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HepG2 human hepatocellular carcinoma cells were obtained from the American Type Culture Collection (ATCC) (Manassas, VA, USA) were cultured with high-glucose Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT, USA), 100 IU/ml penicillin and 100 IU/ml streptomycin (Amresco, Solon, OH, USA) in an atmosphere of 5% CO2 at 37°C.
The HepG2 cells were seeded at a density of 2×105 cells/ml. The cells were divided into five groups: the control group; the oleic acid (OA) group; the OA+RKI-1447 (200 nM) group; the OA+RKI-1447 (400 nM) group; and the OA+RKI-1447 (800 nM) group. Then, 24 h later, the cells were treated with or without RKI-1447 for 2 h, followed by 100 μM oleic acid dissolved in bovine serum albumin (BSA) solution. The control group was treated with BSA at the same volume. After further culture for 24 h, the cells were collected for further study.
The HepG2 cells were seeded at a density of 2×105 cells/ml. The cells were divided into five groups: the control group; the oleic acid (OA) group; the OA+RKI-1447 (200 nM) group; the OA+RKI-1447 (400 nM) group; and the OA+RKI-1447 (800 nM) group. Then, 24 h later, the cells were treated with or without RKI-1447 for 2 h, followed by 100 μM oleic acid dissolved in bovine serum albumin (BSA) solution. The control group was treated with BSA at the same volume. After further culture for 24 h, the cells were collected for further study.
9
HepG2 Hepatocellular Carcinoma Cell Culture
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HepG2 human hepatocellular carcinoma cells (American Type Culture Collection, Manassas, VA, USA) were cultured in Dulbecco’s modified Eagle’s Medium (cellgro®; Mediatech, Inc., Manassas, VA, USA) with 10% fetal bovine serum (FBS; Thermo Fisher Scientific, Rockford, IL, USA). Freshly trypsinized HepG2 cells were suspended at 1×105 cells/ml in standard HepG2 culture medium and seeded at a density of 2×104 cells per well in standard 24-well tissue culture plates. Subsequent to seeding, the cells were incubated at 37°C in a 90% air/10% CO2 atmosphere, and 500 μl fresh medium was supplied every other day to the cultures following removal of the supernatant.
10
Culturing Human Hepatocellular Carcinoma Cells
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Human HepG2 hepatocellular carcinoma cells were obtained from American Type Culture Collection (Manassas, VA, USA). Human hepatocellular carcinoma cell lines BeL7402 were obtained from the Cell Bank of the Chinese Academy of Science (Shanghai, China). Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% FBS, penicillin (100 U/mL), and streptomycin (100 μg/mL) at 37°C in 5% CO2 incubator. Trypsin (0.25%)/EDTA solution was used to detach the cells for subculturing the cells. Insulin was purchased from Sigma (St. Louis, MO).
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