Hct116

Manufactured by Genechem

Sourced in China

The HCT116 is a cell line derived from a human colorectal carcinoma. It is a commonly used model system for studying various aspects of cancer biology and drug development.

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23 protocols using hct116

Generating Stable INPP4B Expression Cell Lines

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SW620 and HCT116 cells (supplied by Shanghai Suer Biotechnology Co., Ltd.) were cultured in DMEM (Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing 10% FBS (Thermo Fisher Scientific) and 100 U/mL penicillin/streptomycin in an incubator with 5% CO₂ at 37°C.
In order to generate SW620 cells with a stable expression of INPP4B, SW620 cells were transfected with the pCMV-2B control vector or pCMV-2B-INPP4B vector with Lipofectamine 3000 (Thermo Fisher Scientific), according to the recommendations of the manufacturer. At 48 hours post-transfection, the transfected cells were selected with 0.5 mg/mL of G418 (Sigma-Aldrich Co., St Louis, MO, USA) for 2 weeks.
For establishing stable INPP4B knockdown HCT116 cells, HCT116 cells were infected with lentiviral particles harboring control shRNA or INPP4B shRNA (GeneChem Co., Shanghai, China). After 48 hours of infection, cells were selected in culture medium containing 1 μg/mL of puromycin (Thermo Fisher Scientific) for 10 days.

Ruan X.H., Liu X.M., Yang Z.X., Zhang S.P., Li Q.Z, & Lin C.S. (2019). INPP4B promotes colorectal cancer cell proliferation by activating mTORC1 signaling and cap-dependent translation. OncoTargets and therapy, 12, 3109-3117.

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Modulating β-catenin Expression in CRC Cells

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For β-catenin knockdown analysis, small interfering RNAs (siRNA) targeting β-catenin were designed and synthesized by Shanghai GenePharma Ltd (Shanghai, China; 5′-GGACACAGCAAUUUGUTT-3′) and transfected into CRC cells (HCT116 and Lovo) using Lipofectamine 3000 transfection reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. Scrambled sequences with no homology to any human gene were used as negative controls. For the overexpression of β-catenin, HCT116 and Lovo cells were transduced with 5 μg of a pcDNA3.1-β-catenin overexpression plasmid (Shanghai GeneChem Co., Ltd, Shanghai, China); cells transfected with the empty pcDNA3.1 vector served as the control. To confirm that the constructs worked, cells were collected 24 h after transfection and β-catenin protein expression levels were detected by Western blot.

Zeng Q., Che Y., Zhang Y., Chen M., Guo Q, & Zhang W. (2020). Thymol Isolated from Thymus vulgaris L. Inhibits Colorectal Cancer Cell Growth and Metastasis by Suppressing the Wnt/β-Catenin Pathway. Drug Design, Development and Therapy, 14, 2535-2547.

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Culturing Human Colorectal Cancer Cell Lines

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The SW620, SW480, HCT116, LOVO, and HT-29 human CRC cell lines as well as NCN460 normal colonic epithelial cells were obtained from Shanghai Genechem Co. Ltd. (Shanghai, China). The CRC cell lines were cultured in L-15 medium (Gibco, Carlsbad, USA), and NCN460 cell lines were grown in RPMI 1640 (Gibco BRL). The medium were supplemented with 10% fetal bovine serum (Gibco, 10099-14) and 1% penicillin-streptomycin (Gibco) and incubated at 37°C and 5% CO₂.

Zhang Z., Tao Y., Hua Q., Cai J., Ye X, & Li H. (2020). SNORA71A Promotes Colorectal Cancer Cell Proliferation, Migration, and Invasion. BioMed Research International, 2020, 8284576.

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Stable AIM2 Overexpression in HCT116 Cells

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The human CRC HCT116 cell line was purchased from Genechem Co. (Shanghai, China) and cultured in RPMI 1640 supplemented with 10% fetal bovine serum, 100 U/mL penicillin and 100 μg/mL streptomycin at 37°C with 5% CO₂. Lentiviral vector containing AIM2 was purchased from Genechem Co. HCT116 cells were infected with lentivirus to generate cell lines with stable expression of AIM2. HCT116 cells were incubated with infection medium containing recombinant lentivirus vectors at a multiplicity of infection of 20 for 16 hours, and then the medium was replaced with fresh complete medium. Green fluorescent protein (GFP) expression was observed by fluorescence microscopy to determine the proportion of GFP-positive cells at 72 hours. Empty vector was used as a negative control. The transfection efficiency was confirmed by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting.

Chen J., Wang Z, & Yu S. (2017). AIM2 regulates viability and apoptosis in human colorectal cancer cells via the PI3K/Akt pathway. OncoTargets and therapy, 10, 811-817.

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Culturing Human Colon Cancer Cells

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HCT116 and HT29 cells, a human colon cancer cell line, were purchased from GeneChem Co., Ltd. (Shanghai, China). The cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum, 100 µg/mL ampicillin, and 100 µg/mL streptomycin, at 37°C with 5% CO2 and 95% air.

Huang S., Zhang Z., Li W., Kong F., Yi P., Huang J., Mao D., Peng W, & Zhang S. (2020). Network Pharmacology-Based Prediction and Verification of the Active Ingredients and Potential Targets of Zuojinwan for Treating Colorectal Cancer. Drug Design, Development and Therapy, 14, 2725-2740.

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Exosome Treatment of Colon Cancer Cells

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Human colon cancer cell lines HCT-116, HT-29 and SW-480 were purchased from Genechem Biotechnology Company (The cell lines of Genechem Biotechnology Company were purchased from ATCC). All the cells were maintained in Dulbecco’s modified Eagle’s medium with 10% FBS (Life Technologies, NY) and 1% antibiotic–antimycotic solution (Life Technologies, NY).
For the exosome treatment, colon cancer cells (HCT-116, HT-29 and SW-480) were treated with 10 μg/ml of BM-MSC-derived exosomes or control PBS q.o.d for 1–2 weeks. For example, at 1, 3, 5, 7, 9.

Li H, & Li F. (2018). Exosomes from BM-MSCs increase the population of CSCs via transfer of miR-142-3p. British Journal of Cancer, 119(6), 744-755.

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Silencing DDX10 in CRC Cell Lines

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Our study used the human CRC cell lines RKO, HCT116, SW480 and LoVo, which were purchased from GeneChem (Shanghai, China). The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; HyClone, Thermo Fisher Scientific) containing 10% foetal bovine serum (FBS; Gibco‐Invitrogen Corp.) at 37 °C with 5% CO₂ in strict accordance with the standard. All lentiviruses used in this research were purchased from GeneChem (Shanghai, China). We transfected short hairpin RNA (shRNA) lentivirus targeting DDX10 and empty vector (controls) into HCT116 cells and RKO cells in strict accordance with the official technical instructions. The DDX10 RNAi sequence was 5′-GATGTGAGCAAGTTACCTATA-3′.

Zhou X., Liu Z., He T., Zhang C., Jiang M., Jin Y., Wu Z., Gu C., Zhang W, & Yang X. (2022). DDX10 promotes the proliferation and metastasis of colorectal cancer cells via splicing RPL35. Cancer Cell International, 22, 58.

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Colorectal Cancer Cell Line Characterization

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The CRC cell lines SW480 and HCT116 were obtained from the GeneChem Company (Shanghai, P.R. China). The CRC cell lines HT29, DLD-1, and RKO and human embryonic kidney (HEK293T) cells were obtained from the State Key Laboratory of Molecular Oncology, Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College. SW480 cells were cultured in DMEM (Thermo Fisher Scientific, Waltham, MA, USA), and the other four cell lines were cultured in RPMI-1640 (Thermo Fisher Scientific) medium supplemented with 100 U/ml penicillin, 100 mg/ml streptomycin (Invitrogen, Waltham, MA, USA), and 10% fetal bovine serum (Biological Industries, Kibbutz Beit Haemek, Israel). All the CRC cells were incubated at 37°C in a 5% CO₂ humidified atmosphere. Cells were transiently transfected with si-FEZF1-AS1 (GenePharma, Shanghai, P.R. China), orthodenticle homeobox 1 (OTX1) overexpression plasmid (Generay, Shanghai, P.R. China), and miR-30a-5p mimics and inhibitors (RiboBio, Guangzhou, P.R. China) using Lipofectamine 2000 (Invitrogen).

Li J., Zhao L.M., Zhang C., Li M., Gao B., Hu X.H., Cao J, & Wang G.Y. (2020). The lncRNA FEZF1-AS1 Promotes the Progression of Colorectal Cancer Through Regulating OTX1 and Targeting miR-30a-5p. Oncology Research, 28(1), 51-63.

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Bacterial Infection Assay in Human Cells

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Fn strain ATCC 25586 was purchased from American Type Culture Collection (ATCC) and grown in Columbia blood agar (Sigma, USA) in an anaerobic bag (Merier, France) at 37 °C as previously described [15 (link)]. HCT116, HT29 and 293 T cells were obtained from GeneChem and cultured in DMEM-F12(Gibco, USA) supplemented with 10% FBS (Gibco) and 1% penicillin and streptomycin (Beyotime, China) at 37 °C in a humidified 5% CO2 atmosphere. For Fn infection assay, cells were cultured in medium without antibiotics and incubated with Fn at a multiplicity of infection (MOI) of 100:1 as previously described [27 (link)].

Zhang S., Yang Y., Weng W., Guo B., Cai G., Ma Y, & Cai S. (2019). Fusobacterium nucleatum promotes chemoresistance to 5-fluorouracil by upregulation of BIRC3 expression in colorectal cancer. Journal of Experimental & Clinical Cancer Research : CR, 38, 14.

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Profiling Human Cancer Cell Lines

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The human colorectal HCT 116 (CVCL_0291), Caco-2(CVCL_0025), HT-29 (CVCL_0320), SW480 (CVCL_0546), SW620 (CVCL_0547), breast MCF-7(CVCL_0031), T-47D (CVCL_0553), MDA-MB-231(CVCL_0062), ovarian Caov-3(CVCL_0231), SK-OV-3(CVCL_0532) cancer cell lines and HEK293T(CVCL_0063) were obtained from the GeneChem Corporation (Shanghai, China). OVCAR-3(CVCL_0465) was obtained from the Nanjing KeyGen Biology (Nanjing, China). All human cell lines have been authenticated using STR profiling. OVCAR-3, Caco-2, MCF-7, Caov-3 and HEK293T cells were cultured in DMEM (Gibco, Carlsbad, CA, USA). T-47D and OVCAR-3 cells were cultured with RPMI 1640 (Gibco, Carlsbad, CA, USA). HCT 116 and HT-29 cells were cultured with McCoy’s 5a (Gibco, Carlsbad, CA, USA). SW480, SW620, and MDA-MB-231 cells were cultured with Leibovitz’s L-15 (Gibco, Carlsbad, CA, USA) medium containing 10% fetal bovine serum, 100 U/mL penicillin, and 100 mg/mL streptomycin. To inhibit NF-κB activities, BAY 117082 (Selleckchem, Houston, TX, USA) was used.

Zhao L., Jiang L., Zhang M., Zhang Q., Guan Q., Li Y., He M., Zhang J, & Wei M. (2021). NF-κB-activated SPRY4-IT1 promotes cancer cell metastasis by downregulating TCEB1 mRNA via Staufen1-mediated mRNA decay. Oncogene, 40(30), 4919-4929.

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