Ls174t

Manufactured by American Type Culture Collection

Sourced in United States, United Kingdom, China

The LS174T is a cell line derived from a human colon adenocarcinoma. It is a widely used model for the study of intestinal epithelial cells and their response to various stimuli.

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LS174T cells (20 citations) LS174T (ATCC CL-188) (6 citations) LS174T cell line (3 citations) LS174T colon cancer cells (3 citations) LS174T cells (CL-188) (2 citations) LS174T CL-188 (2 citations)

303 protocols using ls174t

Colorectal Cancer Cell Culture Conditions

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For the experiments, three different colorectal cancer cell lines were used—HCA-2 (Duke C), LS 174T (Duke B), and SW 1116 (Duke A), along with a normal epithelial cell line known as CCD 841 CoN. All the cell lines were provided by ATCC (American Type Culture Collection ATCC^®, Old Town Manassas, VA, USA). To promote optimal cell growth, we utilized specific culture media for each cell line. For CCD 841CoN and LS 174T cell lines, we used Eagle’s minimum essential medium (EMEM) (ATCC 30-2003), while Dulbecco’s modified Eagle’s medium/Nutrient Mixture F-12 Ham (DMEM) from Sigma-Aldrich D8437 was used for the SW 1116 and HCA-2 cell lines. Additionally, both media were supplemented with 10% fetal bovine serum ((FBS), ATCC 30-2020), and 1% penicillin-streptomycin-neomycin stabilized solution (Sigma-Aldrich P4083) for optimal growth conditions.

Brzozowa-Zasada M., Piecuch A., Bajdak-Rusinek K., Gołąbek K., Michalski M., Matysiak N, & Czuba Z. (2024). A Prognostic Activity of Glutaredoxin 1 Protein (Grx1) in Colon Cancer. International Journal of Molecular Sciences, 25(2), 1007.

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Colon Cancer Cell and Organoid Models

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All cell lines were maintained in a humidified atmosphere with 5% CO₂. HT29 cells were cultured in McCoys 5A media (Corning), NCI-H508 in RPMI 1640 media (Corning) and LS-174T cells in DMEM, supplemented with 10% FBS (Gibco). All cell lines were purchased from the ATCC; LS174T (2020), NCI-H508 (2019), HT29 (verified by STR profiling and tested for Mycoplasma in 2019 by IDEXX). LS174T and NCI-H508 were not further authenticated or tested for mycoplasma because of their recent purchase from ATCC. All cells used in experiments were passaged fewer than 10 times. Normal human organoids derived from the ascending colon (83) were obtained from the Dr. Jason Spence and the University of Michigan Translational Tissue Modeling Laboratory(17 ). Colon cancer organoids were derived from PDX models 519858 162-T (519) and 817829 284-R (817) obtained from the NCI Patient-Derived Models Repository. Mutational status of key genes for these models is provided in Supplemental Table 1. For additional details on treatments and organoid culture see the supplemental methods.

Miller S.A., Policastro R.A., Sriramkumar S., Lai T., Huntington T.D., Ladaika C.A., Kim D., Hao C., Zentner G.E, & O’Hagan H.M. (2021). LSD1 and aberrant DNA methylation mediate persistence of enteroendocrine progenitors that support BRAF mutant colorectal cancer. Cancer research, 81(14), 3791-3805.

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Profiling human CRC cell lines

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Four human CRC cell lines were used in this study. HT‐29, LoVo, and LS174T cell lines were purchased from ATCC. The identity of all cell lines was confirmed by short tandem repeat (STR) testing. STR DNA profiling aids in the identification of human cell lines derived from individual tissue, ensuring the purity of cultures and preventing cross‐contamination. The DLD‐1 cell line was obtained from the Japanese Collection of Research Bioresources (JCRB) Cell Bank (National Institutes ofS Biomedical Innovation, Health and Nutrition). The cell lines were selected based on a previous report describing their histology after establishment as xenografts in nude mice.²⁰ SUIT‐2, a pancreatic cancer cell line without affinity for rBC2LCN,⁴ was also obtained from the JCRB Cell Bank for use as a negative control in each experiment. HT‐29, LoVo, and LS174T were cultured in McCoy’s 5a, F‐12K, and E‐MEM (all ATCC‐formulated media), respectively. DLD‐1 and SUIT‐2 were cultured in RPMI 1640 and D‐MEM (both from FUJIFILM Wako Pure Chemical), respectively. Each medium was supplemented with 10% FBS (Thermo Fisher Scientific) and 1% penicillin‐streptomycin (FUJIFILM Wako Pure Chemical).

Kitaguchi D., Oda T., Enomoto T., Ohara Y., Owada Y., Akashi Y., Furuta T., Yu Y., Kimura S., Kuroda Y., Kurimori K., Miyazaki Y., Furuya K., Shimomura O, & Tateno H. (2020). Lectin drug conjugate therapy for colorectal cancer. Cancer Science, 111(12), 4548-4557.

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Cell Culture Protocol for Colorectal Cancer

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LS174T (Duke’s type B), HT-29 and RKO cell lines were purchased from American Type Culture Collection (ATCC, Rockville, MD, United States). LS174T, HT-29 and RKO cells were grown in Eagle’s Minimum Essential Medium (EMEM) (ATCC 30-2003) or in Dulbecco’s Modified Eagle Medium (DMEM) with high glucose (4.5 g/L) (Thermo scientific), respectively. Culture media were supplemented with 10% (v/v) fetal bovine serum (FBS) (Dutscher, France) and 1% penicillin-streptomycin (v/v, Invitrogen, Cergy-Pontoise, France). Cultures were maintained at 37°C in a humidified atmosphere containing 5% CO2 (v/v). Cells were routinely passaged at preconfluency using 0.05% trypsin, 0.53 mM EDTA (Invitrogen, 25300) and screened for the absence of mycoplasma using PCR methods.

Le C.C., Bennasroune A., Collin G., Hachet C., Lehrter V., Rioult D., Dedieu S., Morjani H, & Appert-Collin A. (2020). LRP-1 Promotes Colon Cancer Cell Proliferation in 3D Collagen Matrices by Mediating DDR1 Endocytosis. Frontiers in Cell and Developmental Biology, 8, 412.

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Cultivation and Characterization of Human Colorectal Adenocarcinoma Cell Lines

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A total of eight human colorectal adenocarcinoma cell lines—SW-480 (CLS Cat# 300302/p716_SW-480, RRID: CVCL_0546), DLD-1 (CLS Cat# 300220/p23208_DLD-1, RRID: CVCL_0248), HCT-15 (CLS Cat# 300229/p23303_HCT-15.html, RRID: CVCL_0292), HCT-116 (CLS Cat# 300195/p19841_HCT116.html, RRID: CVCL_0291), HT-29 (NCI-DTP Cat# HT-29, RRID: CVCL_0320), LS174T (CLS Cat# 300392/p720_LS-174T, RRID: CVCL_1384), SW-620 SW620 (NCI-DTP Cat# SW-620, RRID: CVCL_0547) and LoVo (NCI-DTP Cat# LOVO, RRID: CVCL_0399)—were obtained from the American Type Culture Collection (ATCC, Rockville, Maryland). The cell lines were cultivated at 37 °C in high-glucose Dulbecco’s modified Eagle’s medium (DMEM) containing 10% heat-inactivated fetal bovine serum (FBS), 50 U/mL penicillin, and 50 µg/mL streptomycin (Sigma-Aldrich Chemical Co., USA) in an atmosphere of 5% CO₂ and 95% humidified air. Subculture was performed when 90% confluence was reached. Human cell line identification by short tandem repeat profile testing, according to the American National Standards Institute, has shown an appropriate match for HCT-116 and DLD-1 cell lines.

Alburquerque-González B., Bernabé-García M., Montoro-García S., Bernabé-García Á., Rodrigues P.C., Ruiz Sanz J., López-Calderón F.F., Luque I., Nicolas F.J., Cayuela M.L., Salo T., Pérez-Sánchez H, & Conesa-Zamora P. (2020). New role of the antidepressant imipramine as a Fascin1 inhibitor in colorectal cancer cells. Experimental & Molecular Medicine, 52(2), 281-292.

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In vivo Plasmid Stability Assay

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Plasmid stability and enzymatic tests were performed in 6-week-old female nude mice (NcrNu strain; Taconic) bearing tumors derived from a human colorectal cancer cell line, LS174T [American Type Culture Collection (ATCC)], that harbor a firefly luciferase transgene (LS174T_LucF, Table 1). Tumors were initiated by subcutaneous injection of 5 × 10⁶ cells in 100 µl of PBS per flank and grown for 1 to 2 weeks until they reached a size of 5 to 10 mm. To obtain measures of plasmid stability in vivo, EcN-luxCDABE bacteria with pTKW plasmids were injected intravenously at a dosage of 1 × 10⁶ bacteria, and at the designated time point, tumors were sterilely extracted and homogenized using a tissue dissociator (Miltenyi), an aliquot of which was seeded on each of LB erythromcyin (100 µg/ml), and LB erythromycin + kanamycin plates to obtain the percent of bacteria carrying resistant plasmids, or measured for lacZ activity as mentioned above.

Danino T., Prindle A., Kwong G.A., Skalak M., Li H., Allen K., Hasty J, & Bhatia S.N. (2015). Programmable probiotics for detection of cancer in urine. Science translational medicine, 7(289), 289ra84.

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Culturing Colon Cancer Cell Lines and Primary Colonic Epithelial Cells

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The human colon cancer cell lines Caco‐2, HT‐29, LS174T, SW480 and DLD‐1 (American Type Culture Collection, Rockville, MD, USA) and CDX2‐inducible LS174T cells [9] were cultured in Dulbecco's modified Eagle's medium supplemented with 10% FBS and 1% glutamine and maintained at 37 °C and 5% CO₂. Primary colonic epithelial cells were isolated from four nonpregnant healthy people over the age of 18, by pooling six biopsies from either colon transversum or descendens taken at the same time during routine colonoscopy where all investigations subsequently turned out to be normal. The method was as described previously [10]. The biopsies were collected with informed written consent at the Department of Gastroenterology, Herlev Hospital, Denmark. The study methodologies conformed to the standards set by the Declaration of Helsinki and were approved by the Capital Region of Denmark Committee on Health Research Ethics.

Larsen S., Seidelin J.B., Davidsen J., Dahlgaard K., Nielsen C.H., Bennett E.P., Pedersen O.B., Coskun M, & Troelsen J.T. (2021). CDX2 regulates interleukin‐33 gene expression in intestinal epithelial cells (LS174T). FEBS Open Bio, 11(6), 1638-1644.

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Culturing Human and Mouse Cell Lines

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SKOV-3 (Human ovarian cancer cell), KB (human oral epidermal carcinoma cells), MCF7 (human breast cancer cells), PC-3 (human prostate adenocarcinoma cells), M109 (mouse lung cancer cells), NIH/3T3 (mouse embryonic fibroblasts), peritoneal MΦ (peritoneal macrophages cell line), LS174T (human hepatocyte carcinoma cells), RAW264.7 (murine macrophages) and B16F10 (mouse melanoma cells) were obtained from ATCC (Manassas, VA). HepG2 (human liver carcinoma cells), Huh7 (human hepatocyte carcinoma cells) were donated by Prof. Wanqing Liu (Wayne State University), and TUBO (murine mammary carcinoma cells) was by Prof. Stephen J. Kron (University of Chicago). SKOV-3, MCF7, PC-3, Huh7, KB, HepG2 and TUBO cells were cultured in RPMI 1640 medium, RAW264.7, PM, NIH/3T3, B16F10 and M109 in DMEM medium, and LS174T cells in MEM medium. The media contained 10% fetal bovine serum (FBS) or 10% fetal calf serum, 100 U/mL penicillin and 100 μg/mL streptomycin.

Wang J., Meng F., Kim B.K., Ke X, & Yeo Y. (2019). In-vitro and in-vivo difference in gene delivery by lithocholic acid-polyethyleneimine conjugate. Biomaterials, 217, 119296.

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Activating TGF-β in CRC Cell Lines

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Human CRC cell lines SW480, SW620, HCT116, HT29, DLD1, LoVo and LS174T were acquired from ATCC and cultured under the conditions recommended by the provider. For TGF-β activation, the cells were treated with 10ng/ml TGF-β1 for 96 hours, and TGF-β1 was replaced after 48 hours.

Bu P., Wang L., Chen K.Y., Rakhilin N., Sun J., Closa A., Tung K.L., King S., Varanko A.K., Xu Y., Chen J.H., Zessin A.S., Shealy J., Cummings B., Hsu D., Lipkin S.M., Moreno V., Gümüş Z.H, & Shen X. (2015). miR-1269 Promotes Metastasis and Forms a Positive Feedback Loop with TGF-β. Nature communications, 6, 6879.

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Authenticated Cell Lines for Protein Interaction Studies

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The following authenticated cell lines were purchased from ATCC: MDCK.2 (CRL-2936, RRID:CVCL_B034), HEK293 (CRL-1573, RRID:CVCL_0045), and human colon cancer lines including HT29 (HTB-38, RRID:CVCL_0320), HCT116 (CCL-247, RRID:CVCL_0291), HCT15 (CCL-225, RRID:CVCL_0292), SW480 (CCL-228, RRID:CVCL_0546), Caco2 (HTB-37, RRID:CVCL_0025), SW620 (CCL-227, RRID:CVCL_0547), Ls174T (CL-188, RRID:CVCL_1384), and T84 (CCL-248, RRID:CVCL_0555). Cells were cultured following the manufacturer's guidelines, and passaged up to five times after each thawing. All cell lines were routinely tested for Mycoplasma contamination using Universal Mycoplasma Detection Kit (ATCC, 30–1012K). HEK293 cells were transfected with equimolar amounts of control empty plasmid or plasmid encoding FILIP1L-HA and/or Flag-PFDN1 using lipofectamine 3000 solution (Thermo Fisher Scientific) following the manufacturer's protocols. For MDCK.2 cells, using the 4D-Nucleofector system (Lonza; SE solution; program CM 113), homogenous expression in over 90% of cells was routinely achieved. At 24 to 48 hours following transfection, transfected cells were subjected to downstream assays such as immunoprecipitation, immunoblot, and immunofluorescence staining.

Kwon M., Rubio G., Nolan N., Auteri P., Volmar J.A., Adem A., Javidian P., Zhou Z., Verzi M.P., Pine S.R, & Libutti S.K. (2021). FILIP1L Loss Is a Driver of Aggressive Mucinous Colorectal Adenocarcinoma and Mediates Cytokinesis Defects through PFDN1. Cancer Research, 81(21), 5523-5539.

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