Bt474

Manufactured by American Type Culture Collection

Sourced in United States, Germany, United Kingdom, Japan, Sweden

The BT474 is a cell line derived from a human breast carcinoma. It is a well-characterized model for studying breast cancer biology and is commonly used in research and drug development.

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868 protocols using bt474

Cell Culture and Authentication Protocols

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MCF-10A cells and their derivatives MCF-ErbB2 and MCF-MekDD cells were provided by M. Reginato (Drexel University, USA) [16 (link)]. MCF10A cells were authenticated as published [11 (link)]. Lack of mycoplasma contamination in all cells was established as published [11 (link)]. BT-474 (American Type Culture Collection (ATCC), Manassas, VA, USA), BT474TR and BT474T cells were cultured as published [11 (link)]. Generation of BT-474TR cells is published [17 (link)]. To generate tumorigenic BT474T cells, BT474 cells were implanted orthotopically into the mammary fat pad of severe combined immunodeficiency mouse, and resulting tumors were serially passaged into new hosts over a three-year period [18 (link)]. AU-565 (ATCC), SKBR3 (ATCC) and 293 T cells (provided by A. Stadnyk, Dalhousie University) were cultured as published [11 (link)]. To detach the cells from the ECM, they were plated in suspension culture above a layer of 1% sea plaque agarose polymerized in respective culture medium not containing any additional ingredients.

Liu X., Chipurupalli S., Jiang P., Tavasoli M., Yoo B.H., McPhee M., Mazinani S., Francia G., Kerbel R.S, & Rosen K.V. (2022). ErbB2/Her2-dependent downregulation of a cell death-promoting protein BLNK in breast cancer cells is required for 3D breast tumor growth. Cell Death & Disease, 13(8), 687.

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Generating Drug-Resistant Breast Cancer Cells

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Human BC cell lines MDA-MB-231, MCF-7, BT474, and HCC1937 (parental drug-sensitive cells) provided by American Type Culture Collection (ATCC, Manassas, VA, USA) were exposed to gradient concentrations of different drugs (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany), respectively, to generate cisplatin-resistant BT474 (BT474/Cis) cells, doxorubicin-resistant MCF-7 (MCF-7/Dox) cells, and fulvestrant-resistant HCC1937 (HCC1937/Ful) cells. Cells were cultivated in complete modified Eagle's medium with 10% fetal bovine serum.

Wang B., Wang Y., Wang X., Gu J., Wu W., Wu H., Wang Q, & Zhou D. (2022). Extracellular Vesicles Carrying miR-887-3p Promote Breast Cancer Cell Drug Resistance by Targeting BTBD7 and Activating the Notch1/Hes1 Signaling Pathway. Disease Markers, 2022, 5762686.

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Cell Culture Protocols for Cancer Research

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N87, SKBR3, BT474, and MCF7 cells were obtained from the American Type Culture Collection (ATCC). N87 and SKBR3 cells were cultured in RPMI 1640 (Gibco) with 10% fetal bovine serum (Gibco) and penicillin-streptomycin (100X; Gibco). BT474cells were cultured in Hybri-Care Medium (ATCC) with 10% fetal bovine serum (Gibco) and Penicillin/Streptomycin/Glutamine (100X; Gibco). MCF7 cells were cultured in RPMI 1640 (Gibco) with 10% fetal bovine serum (Gibco) and penicillin-streptomycin (100X; Gibco). Suspension HEK293 Freestyle (293-F, Life Technologies, USA) cells were grown in serum free Freestyle 293 expression media (Gibco) at 37 °C shaken with 110 rpm in 8% CO₂ incubator (Thermo Scientific).

Hou S.C., Chen H.S., Lin H.W., Chao W.T., Chen Y.S., Fu C.Y., Yu C.M., Huang K.F., Wang A.H, & Yang A.S. (2016). High throughput cytotoxicity screening of anti-HER2 immunotoxins conjugated with antibody fragments from phage-displayed synthetic antibody libraries. Scientific Reports, 6, 31878.

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Establishing a Breast Cancer Cell Line Panel

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The BC cell lines, T-47D, MCF7, MDA-MB-231, SK-BR-3 and BT-474, and human normal mammary cells (MCF10A) were purchased from the American Type Culture Collection (ATCC). T-47D cells was maintained in RPMI-1640 (cat. no. 30-2001; ATCC) supplemented with 0.2 U/ml bovine insulin and 10% fetal bovine serum (FBS) (Gibco; Thermo Fisher Scientific, Inc.). MCF7 and MDA-MB-231 cells were maintained in DMEM (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS. SK-BR-3 cells were maintained in McCoy's 5A medium (Gibco; Thermo Fisher Scientific, Inc.). BT-474 cells were maintained in ATCC Hybri-Care Medium (cat. no. 46-X) supplemented with 10% FBS. MF10A cells were maintained in RPMI-1640 supplemented with 10% FBS. All cells were cultured in an incubator at 37°C with 5% CO₂.

Kong S., Liu J., Zhang B., Lv F., Yu Y, & Qin T. (2021). MicroRNA-337-3p impedes breast cancer progression by targeting cyclin-dependent kinase 1. Oncology Letters, 23(1), 15.

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Breast Cancer Cell Line Culture

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Human breast ductal carcinoma cell line BT-474 (American Type Culture Collection (ATCC^®) HTB-20™, Manassas, VA, USA) and human breast gland adenocarcinoma cell lines SK-BR-3 (ATCC^® HTB-30™, Manassas, VA, USA), MCF-7 (AddexBio C0006008, San Diego, CA, USA), and MDA-MB-231 (AddexBio C0006002, San Diego, CA, USA) were cultured as monolayers in vitro. They were fed with media constituted of Hybri-Care (ATCC^® 46-X™) + 0.15% NaHCO₃ (BT-474), McCoy’s 5A (ATCC^® 30-2007™) (SK-BR-3), MEM (Gibco™ 41090028) (MCF-7), and DMEM 4.5 g/L glucose (Gibco™ 31966021) (MDA-MB-231). All media were supplemented with 10% fetal calf serum (CVFSVF00-01, Eurobio, Courtaboeuf, France) and 1% penicillin–streptomycin mix (Gibco™ 15140122). Cells were cultivated at 37 °C under 95% air and 5% CO₂ humidified atmosphere. They were routinely passaged using 0.25% trypsin–EDTA mix (Gibco™ 25200056) and regularly checked for mycoplasma contamination with the MycoAlert™ detection kit (Lonza LT07-218, Basel, Switzerland).

Kiening M, & Lange N. (2022). Enlarging the Scope of 5-Aminolevulinic Acid-Mediated Photodiagnosis towards Breast Cancers. International Journal of Molecular Sciences, 23(23), 14900.

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Cancer Cell Culturing and Xenograft Processing

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MCF7, MDA-MB-453, A-431, H1781, H1819, Calu-3, BT-474, SKBR3, and NCI-N87 cell lines were purchased from the ATCC. MCF7, MDA-MB-453, and A-431 were cultured in DMEM; H1781, H1819, Calu-3, and NCI-N87 in RPMI-1640; BT-474 in ATCC HybriCare Medium with 1.5 g/L sodium bicarbonate; SKBR3 in the ATCC-formulated McCoy’s 5a Medium Modified. To make the complete medium, all media were supplemented with 10% FBS and 1% streptomycin/penicillin. All cells used in this study were periodically tested negative for Mycoplasma using the Mycoplasma Plus PCR Primer Kit (Agilent). All patient-derived cancer cells or tumor spheroids in this study were collected and studied according to the Declaration of Helsinki and DFCI IRBs. DFCI 429 was established from a pleural effusion sample. Next-Generation Sequencing confirmed de novo HER2 amplification. DFCI315 and DFCI359 were characterized to harbor HER2 exon20 V777_G778insGSP and HER2 exon19 755_757LREdelinsRP, respectively (23 (link)). Xenograft tumors were processed for short-term ex vivo as previously described (23 (link)). The spheroids in a range of 40 to 70 μmol/L were resuspended in type I collagen (Corning) and loaded into the DAX-1 3-D cell culture chip (AIM Biotech). After solidification, the media were applied to the outer channels with indicated drugs.

Inoue M., Kobayashi M., Kozaki S., Zimmer A, & Ueda H. (1998). Nociceptin/orphanin FQ-induced nociceptive responses through substance P release from peripheral nerve endings in mice. Proceedings of the National Academy of Sciences of the United States of America, 95(18), 10949-10953.

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Culturing Human Cancer Cell Lines

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Human breast cancer cell lines (MCF7 and BT-474) and a human ovarian carcinoma cell line (ES-2) were obtained from the American Type Culture Collection (ATCC; Manassas, USA). MCF7 cells were cultured in Dulbecco's Modified Eagle's Medium supplemented with 10% fetal bovine serum (FBS), 0.1 mM non-essential amino acids and 1.0 mM sodium pyruvate. BT-474 cells were cultured in ATCC Hybri-Care Medium (ATCC 46-X) supplemented with 10% FBS and 30 ng/mL epidermal growth factor. ES-2 cells were maintained in McCoy's 5a Medium supplemented with 10% FBS. Cells were maintained at 37°C in a humidity-controlled incubator with 5% CO₂. Paclitaxel and doxorubicin were obtained from Sigma-Aldrich (St. Louis, USA).

Liu C.L., Chen M.J., Lin J.C., Lin C.H., Huang W.C., Cheng S.P., Chen S.N, & Chang Y.C. (2019). Doxorubicin Promotes Migration and Invasion of Breast Cancer Cells through the Upregulation of the RhoA/MLC Pathway. Journal of Breast Cancer, 22(2), 185-195.

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Cell Line Maintenance Protocols

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The OvCAR8 cell line was obtained from Dr. Christina Annunziata (National Cancer Institute). Du145, BT474, and H1975 cell lines were purchased from ATCC. OvCAR8, Du145, and H1975 were maintained in RPMI 1640 (ATCC), and BT474 cells were maintained in Hybri-Care Medium (ATCC).

Schardt J.S., Oubaid J.M., Williams S.C., Howard J.L., Aloimonos C.M., Bookstaver M.L., Lamichhane T.N., Sokic S., Liyasova M.S., O’Neill M., Andresson T., Hussain A., Lipkowitz S, & Jay S.M. (2017). Engineered Multivalency Enhances Affibody-Based HER3 Inhibition and Downregulation in Cancer Cells. Molecular pharmaceutics, 14(4), 1047-1056.

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Breast Cancer Cell Lines for Research

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Human TNBC: MDA-MB-231 and BT-549 cells and triple-positive breast cancer (TPBC, ER⁺, PR⁺, HER2 over-expression) BT-474 (cell line used for the counterselections of the evaluated aptamers in Cell-SELEX procedure) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). MDA-MB-231 cisplatin-resistant (MDA-MB231/cis) cells were generated by treating cells with cisplatin chronically as previously described [28 (link)]. MDA-MB-231, BT-549 and MDA-MB231/cis were grown in Roswell Park Memorial Institute-1640 medium (RPMI-1640, Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS, Sigma-Aldrich). BT-474 were grown in HybriCare medium (ATCC, Manassas, VA, USA) supplemented with 10% FBS. All cells were maintained in a 5% CO₂ atmosphere at 37 °C.

Ibarra L.E., Camorani S., Agnello L., Pedone E., Pirone L., Chesta C.A., Palacios R.E., Fedele M, & Cerchia L. (2022). Selective Photo-Assisted Eradication of Triple-Negative Breast Cancer Cells through Aptamer Decoration of Doped Conjugated Polymer Nanoparticles. Pharmaceutics, 14(3), 626.

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Cultivation of Breast Cell Lines

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The human immortalized breast epithelial cell line (MCF-10A) and five breast cancer cell lines (BT-474, MCF-7, MDA-MB-231, SKBR3 and T-47D) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). MEGMTM Mammary Epithelial Cell Growth Medium BulletKitTM (Lonza/Clonetics Corporation, Walkersville, MD, USA), supplemented with 100 ng/mL cholera toxin, was used for MCF-10A cell culture. BT-474 cells were grown in Hybri-Care Medium (ATCC) containing 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and 1% penicillin/streptomycin (Gibco; Thermo Fisher Scientific, Inc.). MCF-7, MDA-MB-231, SKBR3 and T-47D cell lines were, respectively, maintained in Eagle’s Minimum Essential Medium (ATCC), Leibovitz’s L-15 Medium (Gibco; Thermo Fisher Scientific, Inc.), McCoy’s 5a Medium (Gibco; Thermo Fisher Scientific, Inc.), and RPMI-1640 Medium (Gibco; Thermo Fisher Scientific, Inc.). The other culture conditions were the same as that of BT-474 cells. All cells were kept at 37°C in an incubator in a saturated humidified atmosphere supplied with 5% CO₂.

Yu H., Dong L., Wang H., Zhang Y., Wang Z., Wang C., Xia H, & Bao H. (2021). LINC00504 Promotes the Malignant Biological Behavior of Breast Cancer Cells by Upregulating HMGB3 via Decoying MicroRNA-876-3p. Cancer Management and Research, 13, 1803-1815.

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