HCT-116 and LS-174T cell lines were obtained from ATCC (Manassas, VA) in 2014. Parental LS-174T (ATCC), LS-174T/vector, LS-174T/shPPARδ, LS-174T/shNanog, parental HCT-116 (ATCC), HCT-116/WT, HCT-116/PPARd−/−, HCT-116/vector, and HCT-116/shNanog cells were maintained in McCoy’s 5A medium (Life Technologies) with 10% fetal bovine serum (FBS) (Hyclone, Logan, UT). All CRC cells are used between passages 2 to 5. All cell lines have been tested by MycoProbe Mycoplasma Detection Kit (R&D) and also authenticated before the experiment according ATCC STR database. For GW501516 treatment, the cells were cultured with serum-free medium for 24 or 48 h and then treated with vehicle or indicated concentration of GW501516 for 24 h. After treatment, the cells were subjected to in vitro sphere-forming assay, subcutaneous (sub-Q) injection of NSG mice, q-PCR, Western blotting, luciferase assay, and ChIP assay.
Hct116
Manufactured by Thermo Fisher Scientific
Sourced in United States, China, United Kingdom, Germany, Japan, Austria, Spain
The HCT116 is a cell line derived from a human colorectal carcinoma. It is commonly used in cancer research and drug discovery studies.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (352 mentions)
Hct116, by American Type Culture Collection (332 mentions)
Sw480, by Thermo Fisher Scientific (192 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (188 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (173 mentions)
Sw480, by American Type Culture Collection (144 mentions)
Sw620, by Thermo Fisher Scientific (133 mentions)
Ht 29, by Thermo Fisher Scientific (115 mentions)
Mccoy s 5a medium, by Thermo Fisher Scientific (114 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (97 mentions)
Rpmi 1640, by Thermo Fisher Scientific (95 mentions)
Sw620, by American Type Culture Collection (90 mentions)
Penicillin, by Thermo Fisher Scientific (81 mentions)
Streptomycin, by Thermo Fisher Scientific (80 mentions)
Dld 1, by Thermo Fisher Scientific (69 mentions)
Caco 2, by Thermo Fisher Scientific (56 mentions)
Dld 1, by American Type Culture Collection (55 mentions)
Hek293t, by Thermo Fisher Scientific (44 mentions)
Ncm460, by Thermo Fisher Scientific (44 mentions)
Dmem medium, by Thermo Fisher Scientific (43 mentions)
696 protocols using hct116
1
Colorectal Cancer Cell Lines Characterization
2
Generating HCT116 Cell Line Variants
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The human, male colon carcinoma cell line HCT116 was obtained from the American Type Culture Collection (ATCC). HCT116 Flp-In™ T-REx™ derivatives were generated using the Flp-In™ T-REx™ System (Invitrogen) according to manufacturer instructions. HCT116 TP53−/− cells generated by rAAV-mediated homologous recombination were provided by Bert Vogelstein (Bunz et al., 2002 (link)). To create the HCT116 Flp-In™ T-REx™ GFP-H2B cell line, an open reading frame encoding histone H2B was generated by RT-PCR amplification (Invitrogen) of mRNA prepared from HCT116 cells and cloned as a GFP-tagged fusion into a pcDNA5/FRT/TO-based expression vector (Invitrogen). This plasmid was transfected into the HCT116 Flp-In™ T-REx™ cell line using Lipofectamine Plus (Invitrogen), following manufacturer’s instructions. Cell lines were cultured in DMEM plus 10% fetal calf serum (Life Technologies), 100 U/ml penicillin, 100 U/ml streptomycin and 2 mM glutamine (all from Sigma), then maintained at 37°C in a humidified 5% CO2 atmosphere. All cell lines were authenticated by the Molecular Biology Core Facility at the CRUK Manchester Institute using Promega Powerplex 21 System and periodically tested for mycoplasma.
3
Culturing Colorectal Cancer Cell Lines
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All cell lines (Caco-2, HT29, SW480, DLD1, HCT116, LS174T, RKO) were cultured in a humidified atmosphere (37°C, 5% CO2) in their corresponding medium supplemented with 10% fetal calf serum (Life Technologies) and 1% Glutamin (GE Healthcare). Caco-2, LS174T, RKO and SW480 were cultured in DMEM, high glucose, DLD1 in RPMI, HCT116 and HT29 in McCoys 5A medium (all Life Technologies). All CRC cell lines were screened for mycoplasma contamination using Venor GeM Classic Mycoplasma Detection Kit (Minerva biolabs) and DNA fingerprinting was performed for all seven cell lines at the Leibniz Institute DSMZ, Braunschweig, Germany.
4
Cell Culture and Genetic Manipulation
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All cells were grown in DMEM medium with 10% fetal bovine serum supplemented with non-essential amino acids, l -glutamine, 100 U/ml penicilin and 0.1 mg/ml streptomycin. Cell lines used: hTERT-RPE parental and CSB knockout (12 (link)), Phoenix (14 (link)), U2OS (19 (link)) (ATCC), U2OS-265 (20 (link)) (a kind gift from Roger Greenberg, University of Pennsylvania), U2OS-265-CSB knockout (KO) (11 (link)), 293T (ATCC), HCT116 (Life Technology), HCT116-CSB-KO (11 (link)), AID-DIvA-U2OS (21 (link)) (a generous gift from Gaëlle Legube, University of Toulouse). Parental cells were examined for mycoplasma contamination. Retroviral gene delivery was carried out as described (22 (link),23 (link)) to generate stable cell lines. DNA and siRNA transfections were carried out with respective JetPrime® transfection reagent (Polyplus) and Lipofectamine RNAiMax (Invitrogen) according to their respective manufacturer's instructions.
To induce expression of FokI, U2OS-265 or U2OS-265-CSB-KO cells were treated with both 1 μM Shield-1 (CheminPharm) and 1 μM 4-hydroxytamoxifen (4-OHT, Abcam) for 4 h.
To induce expression of FokI, U2OS-265 or U2OS-265-CSB-KO cells were treated with both 1 μM Shield-1 (CheminPharm) and 1 μM 4-hydroxytamoxifen (4-OHT, Abcam) for 4 h.
5
Regulation of Colorectal Cancer Cell Lines
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The human colorectal cancer cell lines SW480, WiDr, HT-29, and HCT116 were obtained from the American Type Culture Collection (Rockville, MD). The cell lines HT29 and HCT116 were seeded at a density of 1 × 106/ml in RPMI 1640 medium (Gibco, MA), and SW480 and WiDr cells were seeded a density of 1 × 106/ml in Dulbecco’s modified Eagle medium. Both media were supplemented with 10% FBS (CSL Ltd., Australia), 100 IU/ml penicillin, and 50 mg/ml streptomycin sulfate. HT29 and HCT116 cells with low HOXB9 expression were infected with HOXB9-expressing lentiviral constructs (Invitrogen). For the Tet-regulated system, the entry vector was transferred to the lentiviral pLenti4/TO/V5-DEST vector and used in combination with the Tet-repressor vector pLenti6/TR (Invitrogen) according to the manufacturer’s instructions. The induction of HOXB9 was regulated by the addition of 1 μg/ml tetracycline (Invitrogen). To knock down HOXB9 expression, viral constructs of shRNA were generated, and SW480 and WiDr cells were infected as described previously [5 (link)].
6
Cancer Cell Lines Maintenance and Treatment
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The human colorectal cancer lines HCT116 and SW480 and the human lung cancer cell lines H460 and H1299 came from America Type Culture Collection (Manassas, VA). The HCT116 (p53−/−) cell line was a gift of Dr. Bert Vogelstein, Johns Hopkins University. All cell lines were maintained at 37°C and 5% CO2. Cell culture media included McCoy's 5A (Invitrogen, Carlsbad, CA) for HCT116, HCT116 (p53−/−), and SW480 and RPMI-1640 (Invitrogen) for H460 and H1299. The cell culture media were supplemented with 10% fetal bovine serum (HyClone, Logan, UT), 100 units/ml penicillin, 100 μg/ml streptomycin, and 250 ng/ml amphotericin B (Mediatech, Hemdon, VA). Transfection was performed with Lipofectamine 2000 (Invitrogen) following the instructions of the manufacturer. The anticancer drugs used in the study, i.e., 5-fluorouracil (5-FU, 50 μg/ml) and doxorubicin hydrochloride (DOX, 0.4 μg/ml), were purchased from Sigma (ST. Louis, MO). All drugs were dissolved in DMSO and diluted to appropriate concentrations with cell culture media.
7
Establishing Colon Cancer Cell Lines
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The human colon carcinoma cell line HCT116 was obtained from the American Type Culture Collection (ATCC). Human colon carcinoma DLD1, HT29, colo201, colo320, SW620 and human normal colon CCD-33Co cells were gifts from Dr. Keith Bonham in the Saskatoon Cancer Center. HCT116, DLD1, HT29, and SW620 cells were cultured in Dulbecco’s minimum essential medium (DMEM) (Sigma); CCD-33Co cells were cultured in Eagle’s minimum essential medium (EMEM); colo201 and colo320 cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium. All media were supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin (Invitrogen). All cells were cultured in a 5% CO2 atmosphere at 37°C. HCT116-TR stable cell lines were created by transfecting HCT116 cell lines with pLenti6-TR-lentivirus (Invitrogen) and selecting with 10 μg/ml blasticidin (Invitrogen).
8
Colon Cancer Cell Line Characterization
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Two colon carcinoma cell lines (HCT116 and SW620) were purchased from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences (Shanghai, China). Cells were maintained at 37 °C in a humidified air atmosphere containing 5% carbon dioxide in McCOY’s 5A medium (HCT116) or RPMI-1640 (SW620) supplemented with 10% FBS (Invitrogen). The human colon cancer cell lines were recently authenticated by Genetic Testing Biotechnology Corporation (Suzhou, China) using short tandem repeat (STR) profiling. All lines were found to be negative for mycoplasma contamination. SiRNA against PRMT1 and SMARCA4 were synthesized by Jima, China. The sequences used were as follows: PRMT1 siRNA-1, 5′-CUGAAGUCCAGGUCGAUGGUGAAGUC-3′; PRMT1 siRNA-2, 5′-UUGUAGUCUUUGUACUGCC-3′; SMARCA4 siRNA-1, 5′-UCGCUUUGGUUCGCAAAUC-3′; SMARCA4 siRNA-2, 5′-UUCCUCCUCAUUCAGGUCC-3′. HCT116 and SW620 cells were transfected with oligonucleotides or the indicated constructs using Lipofectamine 3000 (Invitrogen) according to the manufacturer’s instructions.
9
Development and Characterization of 5-FU Resistant Colon Cancer Cell Lines
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The human colon cancer cell lines RKO, SW480, HCT116, and LoVo were obtained from the Type Culture Collection of the Chinese Academy of Sciences. All cell lines were cultured as described previously.36 (link) 5-FU-resistant colon cancer cell lines (RKO-5Fures, SW480-Fures, HCT116-5Fures, and LoVo-5Fures) were developed through a stepwise incremental treatment with 5-FU. 5-FU (Sigma-Aldrich) was dissolved in DMSO (Sigma-Aldrich) and stored in airtight containers protected from light before experiments. The parental cell lines RKO, SW480, HCT116, and LoVo were exposed to increasing concentrations of 5-FU stepwise, starting at 1 μM and ending at 100 μM. 5-FU (1 μM) was included in the culture medium for RKO-5Fures, SW480-5Fures, HCT116-5Fures, and LoVo-5Fures to maintain drug resistance. The cells were maintained in 5-FU-free medium at least 2 weeks before the experiments. Cell lines with stable downregulation of miR-1290 were established by lentiviral transduction using a pCDH plasmid (System Biosciences) carrying miR-1290 inhibitor. All cell lines were cultured at 37°C in a humidified atmosphere with 5% CO2 and maintained in DMEM supplemented with 10% fetal bovine serum (Gibco). SW480, HCT116, SW480-5Fures, and HCT116-5Fures were then transfected with the plasmids using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol.
10
Colon Cancer Cell Culture and miR-1180-3p Transfection
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Human normal colon epithelial cells FHC and CRC cells (HCT116, Caco2, LoVo, SW480, SW620) were all obtained from American Type Culture Collection (USA). SW480 and SW620 cells were cultured in L-15 Medium (Gibco, USA) supplemented with 10% fetal bovine serum (FBS, Sigma-Aldrich, USA) in an atmosphere of 100% air. FHC, HCT116, Caco2, and LoVo cells were cultured in RPMI-1640 cell culture medium (Gibco, USA) supplemented with 10% FBS (Sigma-Aldrich, USA) in a humidified incubator containing 5% CO2.
HCT 116 and SW620 cells were subjected to transfection. They were seeded in six-well plates to achieve about 80% of the cell confluence. The sequences of mature miR-1180-3p (miR-1180-3p mimic) and negative control of mimic (NC-mimic) were synthesized and purified by Beijing Generaybiotech (Beijing, China). miR-1180-3p mimic or NC-mimic was transfected into HCT 116 and SW620 cells using Lipofectamine 3000 reagent (Invitrogen, USA) as per the instructions.
RNA from cells was extracted via Trizol (Invitrogen) following the user guide from the manufacturer.
HCT 116 and SW620 cells were subjected to transfection. They were seeded in six-well plates to achieve about 80% of the cell confluence. The sequences of mature miR-1180-3p (miR-1180-3p mimic) and negative control of mimic (NC-mimic) were synthesized and purified by Beijing Generaybiotech (Beijing, China). miR-1180-3p mimic or NC-mimic was transfected into HCT 116 and SW620 cells using Lipofectamine 3000 reagent (Invitrogen, USA) as per the instructions.
RNA from cells was extracted via Trizol (Invitrogen) following the user guide from the manufacturer.
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