The human liver cancer cell line HepG2 and the mouse fibroblast noncancerous cell line L929 were acquired from the National Centre for Cell Sciences (NCCS), Pune. The cell lines were later propagated in Dulbecco's Modified Eagle Medium (DMEM), supplemented with 10% (v/v) fetal bovine serum (FBS), an antimycotic antibiotic, and maintained with a continuous supply of 5% CO2 at 37°C.
Hepg2
Manufactured by National Centre for Cell Science
Sourced in India
HepG2 is a cell line derived from human hepatocellular carcinoma. It is a commonly used in vitro model for the study of liver function and metabolism.
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Lab products found in correlation
Mcf 7, by National Centre for Cell Science (8 mentions)
Phosphoethanolamine, by Merck Group (2 mentions)
Hep3b, by National Centre for Cell Science (2 mentions)
Wrl 68, by National Centre for Cell Science (2 mentions)
Ht 29, by National Centre for Cell Science (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Large vessel endothelial supplement, by Thermo Fisher Scientific (1 mentions)
Human recombinant egf, by Corning (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin antibiotic, by HiMedia (1 mentions)
Dulbecco modified eagle medium (dmem), by HiMedia (1 mentions)
Fetal bovine serum (fbs), by HiMedia (1 mentions)
Hek 293, by National Centre for Cell Science (1 mentions)
Nci h460, by National Centre for Cell Science (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Me 180, by National Centre for Cell Science (1 mentions)
Caco 2, by National Centre for Cell Science (1 mentions)
Wi 38, by National Centre for Cell Science (1 mentions)
Raw 264, by National Centre for Cell Science (1 mentions)
32 protocols using hepg2
1
Culturing HepG2 and L929 Cell Lines
2
Culturing Human Liver and Endothelial Cells
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Human HCC cell lines HepG2, Huh7, and Hep3Bwere purchased from National Centre for Cell Science (NCCS), Pune, India was cultured in DMEM with 10% FBS and 1% Penicillin Streptomycin (Invitrogen, Carlsbad, CA, USA). Normal adult liver epithelial cells (THLE-2: ATCC-CRL-2706) were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA), cultured in bronchial epithelial cell growth medium (BMEM) supplemented with 0.08% phosphoethanolamine (Sigma Aldrich Co, St. Louis, MO, USA), 0.06% human recombinant EGF (Corning Inc., Corning, NY, USA) and 10% FBS. Human umbilical endothelial cells (HUVEC) were purchased from Thermo Fisher Scientific (Waltham, MA, USA) and cultured in medium 200 basal media with large vessel endothelial supplement (Gibco). All the cells were cultured in a humidified chamber with 5% CO2 at 37 °C.
3
Culturing and Propagating HepG2 Cells
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Human hepatoma cell line HepG2 was obtained from National Centre for Cell Sciences (NCCS), Pune, India. Cells were cultured under standard cell culture conditions at 37 °C in a controlled humidified atmosphere with 5% CO2 supply and 100% humidity in CO2 incubator (NuAire NU-5830, USA). Culture was done in 25cm2T- flask (Nunc, Roskilde, Denmark) in Dulbecco’s Modified Eagle's medium (DMEM) enriched with nutrients such as glucose, sodium bicarbonate, sodium pyruvate,4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer supplemented with 10% (v/v) heat inactivated Fetal Bovine Serum (FBS), 1% (v/v) Penicillin–Streptomycin 10,000 U/mL solution (all from Invitrogen, San Diego, CA, USA). The cells were then collected from the flask using 0.25% trypsin (Gibco-BRL, Grand Island, NY, USA) and 1 mmol/L EDTA, and then revived for subsequent investigations. In the drug treatment experiments, cells with passage numbers of 2 to 15 were employed.
4
Cell Culture Maintenance Protocol
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HeLa (Human cervical adenocarcinoma), A431 (skin cancer cells), HepG2 (human liver cancer cell line), MCF-7 (breast cancer cells), and HEK (human embryonic kidney) cell lines were procured from the National Centre for Cell Science (NCCS), Pune. They were maintained in Dulbecco’s modified eagle’s medium supplemented with 10% fetal bovine serum, penicillin (100 IU ml-1), and streptomycin (100 IU ml-1) in a humidified 5% CO2 atmosphere at 37°C for experiments.
5
Fructose-Induced Metabolic Alterations in HepG2 Cells
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The human hepatocellular carcinoma cell line (HepG2) was sourced from National Centre for Cell Sciences (NCCS, Pune, India) and grown under standard aseptic conditions using sterile Dulbecco’s Modified Eagle Medium (DMEM; HiMedia Laboratories, India), supplemented with FBS (12%) and Penicillin-Streptomycin Antibiotic (1%, HiMedia Laboratories, India) at 37°C under humidified CO2 (5%) (Shel lab, USA). The cells were seeded aseptically (1x105 cells/2mL well) and allowed to grow for 48 h, either in DMEM (NC), DMEM + 0.55 mM fructose (FC1), DMEM + 1mM fructose (FC2) or DMEM + 1 mM fructose + 0.1 µM Insulin (FC3). The HepG2 cells (FC1-FC3) were exposed to either PG-HM, its fractions (PG-H, PG-C, PG-EA, PG-B, PG-A at 35 µg mL−1); or DMSO (0.1% v/v, VC1–VC3). The supernatant and cell lysates were collected and preserved at −80°C for further analysis of glycogen, carbohydrate metabolizing enzymes (hexokinase, aldehyde dehydrogenase, ketohexokinase, phosphofructokinase), secondary messenger of insulin signaling (PI3K p-tyr-STAT-3, mTOR), hypoxia and inflammation (HIF-1α, VEGF, TNF-α), using commercially available kits in accordance with the manufacturer’s instructions.
6
MTT Assay for Cancerous Cell Lines
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The effect of LFE on cancerous cell lines was measured using a known MTT-assay protocol as described by Denizot & Lang [26 (link)] but with minor modifications. Two different cancerous cells, human breast adenocarcinoma cell line (MCF 7) and human hepatocarcinome cell line (HepG2) were obtained from National Centre for Cell Science, Pune, India. Both the cell lines were treated with different concentrations of LFE in this study.
7
Culturing Hep-G2 Liver Cancer Cells
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Human liver cancer cell line (Hep-G2) was obtained from National Centre for Cell Science (NCCS), Pune and grown in Eagles Minimum Essential Medium containing 10% fetal bovine serum (FBS)22 (link). The cell lines were cultured and incubated according to the procedure given and used for further toxicity studies.
8
Cytotoxicity Assay of Cell Lines
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Fetal bovine serum was obtained from Sigma Aldrich. All the solvents, chemicals, and reagents were purchased from Hi Media laboratories (Mumbai). Cancer cell lines viz., MCF 7 (human breast cancer), HEP G2 (liver cancer), NCIH 460 (non-small cell lung cancer), and non-cancerous human kidney cell line, HEK293 were procured from the National Centre for Cell Sciences (NCCS), Pune.
9
Cytotoxicity Evaluation of Botanical Extracts
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Authenticated cell lines ME-180, HeLa and HepG2 were procured from National Centre for Cell Science, Pune, India. The cells were grown in Roswell Park Memorial Institute-1640, Eagle’s Minimal Essential Medium and Dulbecco’s Modified Eagle Medium media, respectively, and 10% FBS (16000044, Thermo Fisher, Waltham, MA, USA) and 1% antibiotic solution were used for supplementation. Cells were grown in T-25 flasks and were passaged upon confluence using trypsin-EDTA [16 ]. Nearly 5000 cells were seeded per well in 96-well plate and incubated at 37 °C in 5% CO2 incubator and left overnight to enable surface attachment. Cells were treated with extracts (methanol, ethanol and aqueous) with concentrations of 50, 25, 10, 5, 1, 0.5, 0.1, 0.05 mg/mL and left overnight in incubator. 5 mg/mL of MTT per well was added and incubated for 2 h at 37 °C. Formazan crystals were solubilized with 100 μL DMSO and incubated for 10 min. The absorbance was measured at 570 nm and reference at 630 nm.
10
PBMC Isolation and Cancer Cell Lines
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Human blood sample for obtaining primary peripheral blood mononuclear cells (PBMCs) was collected voluntarily in the Health Centre of Tezpur University, Assam, India. The embryonic human liver cancer cell line (HepG2), epithelial colorectal adenocarcinoma cell line (CaCo-2) and epithelial breast cancer cell line (MCF-7) were obtained from National Centre for Cell Science (NCCS), Pune, India. The media for animal cell culture were purchased from HiMedia, India and the chemicals and solvents were obtained from Sigma Aldrich, USA and Merck, Germany.
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