Zr 75 1

Manufactured by American Type Culture Collection

Sourced in United States, Italy

The ZR-75-1 is a cell line derived from a breast carcinoma. It is a standard cell line used in research for the study of breast cancer.

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312 protocols using zr 75 1

Culturing Human Breast Cancer Cell Lines

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Human breast cancer cell lines of MCF7 (ATCC, HTB-22), ZR75-1 (ATCC, CRL-1500), and MDA-MB-231 (ATCC, HTB-26) were obtained from the American Type Culture Collection (Manassas, VA, USA) and sub-cultured, as per supplier’s instructions. MCF7 cells were cultured in Eagle’s Minimum Essential Medium (EMEM) (ATCC) supplemented with 10% fetal bovine serum (FBS) (Corning, Corning, NY, USA), and Human Insulin (Invitrogen, Carlsbad, CA, USA). ZR75-1 cells were cultured in the RPMI-1640 Medium (ATCC, 30-2001), with 10% FBS, and the MDA-MB-231 cells were cultured in the DMEM/F-12 (1:1) medium (Mediatech, Herndon, VA, USA), containing 5% FBS, 4 mM glutamine, 50 µM β-Mercaptoethanol, and 1 mM sodium pyruvate. Cell lines were maintained at 37 °C with 5% CO₂. For the suspension culture, cells were seeded into Corning Ultra-Low Attachment plates, which inhibits immobilization to the surface. Cells were maintained in suspension with media changes every other day, for up to seven days.

Twomey J.D, & Zhang B. (2019). Circulating Tumor Cells Develop Resistance to TRAIL-Induced Apoptosis Through Autophagic Removal of Death Receptor 5: Evidence from an In Vitro Model. Cancers, 11(1), 94.

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Cell Culture Protocols for Cancer Cell Lines

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Cell lines MCF-7, SKBR-3, MDA-MB-231, and ZR-75-1 were obtained from the American Type Culture Collection (ATCC) and grown as follows: MCF-7 using Dulbecco modified Eagle medium (DMEM), SKBR-3 using McCoy’s 5A medium, MDA-MB-231 using Leibovitz’s L-15 medium, and ZR-75-1 using Roswell Park Memorial Institute (RPMI) medium. All media were supplemented with 10 % fetal bovine serum (FBS), 100 U/ml penicillin, 100 μg/ml streptomycin, and 1 % GlutaMAX (Ambion, Calsbad, CA, USA).

Spanheimer P.M., Lorenzen A.W., De Andrade J.P., Kulak M.V., Carr J.C., Woodfield G.W., Sugg S.L, & Weigel R.J. (2015). Receptor Tyrosine Kinase Expression Predicts Response to Sunitinib in Breast Cancer. Annals of surgical oncology, 22(13), 4287-4294.

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Breast Cancer Cell Line Cultivation and Treatments

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Breast cancer cells of MCF-7 and ZR75-1 cell lines were purchased from ATCC. HEK293T was purchased from the Cell Bank of the Chinese Academy of Sciences. All cells were maintained in DMEM medium supplemented with 10% FBS (BI, Biological Industries), 1% Penicillin-Streptomycin Solution (Hyclone) at 37°C in a humidified incubator with 5% CO2. Cell were transferred into special medium (phenol red free) added with charcoal-dextran treated serum before estradiol treatment. For sphere formation assay, no serum was added, as was introduced below. MiR-129 mimics/inhibitors, Let-7b mimics/inhibitors, miR-129 lentiviral based RFP-plasmin were synthesized by GenePharma. Estradiol (estrogen, E1024 Sigma-Aldrich), 5-Fluorouracil (F6627S Sigma-Aldrich), γ-secretase inhibitor (SCP0004 Sigma-Aldrich) were purchased and stored in the central laboratory. Forty-two pairs clinical samples were collected from January 2011 to January 2015 (Supplementary Table 1), and written informed consents were obtained and filed. The medical ethics committee of the institution of Xi’an Jiaotong University approved this study.

Xiao G., Li X., Li G., Zhang B., Xu C., Qin S., Du N., Wang J., Tang S.C., Zhang J., Ren H., Chen K, & Sun X. (2017). MiR-129 blocks estrogen induction of NOTCH signaling activity in breast cancer stem-like cells. Oncotarget, 8(61), 103261-103273.

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Cell Culture of Human Cancer Cell Lines

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Human colon adenocarcinoma cell line HT-29 (Catalogue Number 91072201, ATCC HTB-38) and human breast carcinoma cell line ZR-75-1 (Catalogue Number 87012601, ATCC CRL 1500) were acquired from the European Collection of Authenticated Cell Cultures (ECCAC). In accordance with ECCAC instructions, HT-29 cells were cultured in McCoy’s 5a medium (Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS) (Sigma Aldrich), 2 mM of L-Glutamine (Sigma-Aldrich), 100 U/ml of penicillin and 100 μg/ml of streptomycin (Sigma-Aldrich) at 37°C and 5% CO₂. Cells were passaged when 80–90% confluence was reached, and the media was changed every 2–3 days. In accordance with ECCAC instructions, ZR-75-1 cells were cultured in RPMI 1640 (Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS) (Sigma-Aldrich), 1 mM sodium pyruvate (Sigma-Aldrich), 2 mM of L-Glutamine (Sigma-Aldrich) and 100 U/ml of penicillin and 100 μg/ml of streptomycin (Sigma-Aldrich) at 37°C and 5% CO₂. Cells were passaged when 70–80% confluence was reached, and the media was changed every 2–3 days.

Salguero-Aranda C., Sancho-Mensat D., Canals-Lorente B., Sultan S., Reginald A, & Chapman L. (2019). STAT6 knockdown using multiple siRNA sequences inhibits proliferation and induces apoptosis of human colorectal and breast cancer cell lines. PLoS ONE, 14(5), e0207558.

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Cell Line Maintenance and Reagents

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AU565, HCC1419, NCI-H2170, HCC202, HCC1954, NCI-N87, ZR75-1, SKOV3, ZR75-30, MDAMB175VII, CALU3, MDAMB453, MDAMB361, JIMT1, SKBR3 and HCC2218 cells were obtained from ATCC. OE19 and OE33 were obtained from ECCC; COLO-678 was obtained from DSMZ, and KYSE-410 from Sigma-Aldrich. BT-474-M3 cells (hereafter simply referred to as BT-474) were obtained from Hermes biosciences. All cell lines were maintained in RPMI supplemented with 10% FBS, penicillin, and streptomycin. GSK-1120212 and MK-2206 were purchased from Selleckchem. Recombinant human HRG-β1 (EGF domain) was from R&D Systems.

Kirouac D.C., Du J., Lahdenranta J., Onsum M.D., Nielsen U.B., Schoeberl B, & McDonagh C.F. (2016). HER2+ Cancer Cell Dependence on PI3K vs. MAPK Signaling Axes Is Determined by Expression of EGFR, ERBB3 and CDKN1B. PLoS Computational Biology, 12(4), e1004827.

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Cell Line Maintenance and Compounds

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MCF7 (ATCC), T47D (ATCC), ZR-75-1 (ATCC) cells were maintained in RPMI 1640. Ishikawa (Sigma), HEK293T (ATCC) and MDA-MB-231 (ATCC) cells were maintained in DMEM. CV1 (ATCC) and HEPG2 (ATCC) cells were maintained in MEM. All medium was supplemented with 10% fetal bovine serum (FBS) (Hyclone), 1 mM sodium pyruvate and 1X non-essential amino acids unless otherwise indicated. Unless indicated, tissue culture supplements and medium were purchased from Mediatech or Invitrogen. GDC-0810, arzoxifene and bazedoxifene were synthesized at Seragon Pharmaceuticals or Genentech. Fulvestrant and raloxifene were purchased from Waterstone Technology LLC. 4OH-tamoxifen, endoxifen, and 17β-estradiol were purchased from Sigma Aldrich. Lasofoxifene was purchased from Toronto Research Chemicals.

Joseph J.D., Darimont B., Zhou W., Arrazate A., Young A., Ingalla E., Walter K., Blake R.A., Nonomiya J., Guan Z., Kategaya L., Govek S.P., Lai A.G., Kahraman M., Brigham D., Sensintaffar J., Lu N., Shao G., Qian J., Grillot K., Moon M., Prudente R., Bischoff E., Lee K.J., Bonnefous C., Douglas K.L., Julien J.D., Nagasawa J.Y., Aparicio A., Kaufman J., Haley B., Giltnane J.M., Wertz I.E., Lackner M.R., Nannini M.A., Sampath D., Schwarz L., Manning H.C., Tantawy M.N., Arteaga C.L., Heyman R.A., Rix P.J., Friedman L., Smith N.D., Metcalfe C, & Hager J.H. (2016). The selective estrogen receptor downregulator GDC-0810 is efficacious in diverse models of ER+ breast cancer. eLife, 5, e15828.

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Establishing Validated Breast Cancer Cell Lines

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T47D, MCF7, AU565, MDA-MB-231, MDA-MB-361, and ZR-75-1 cell lines were purchased from ATCC (American Type Culture Collection) in 2011. 4T1 cells were purchased from ATCC in 2013. SUM-159 were obtained from Dr. Jeffrey A. Frost (University of Texas Health Science Center at Houston), human mammary epithelial (HMLE) and HMLE ER-Twist cells were kindly provided by Dr. Sendurai A. Mani (University of Texas MD Anderson Cancer Center) in 2013. All cell lines used in this study were used at low passage and shown to be free of mycoplasma (MycoAlert Mycoplasma Detection Kit, Lonza #LT37-618). Except for SUM-159, all cell lines were obtained either from ATCC or the original source (HMLE). We did no further authentication on the SUM-159 cell line.
Additional procedures are described in Supplementary Materials and Methods.

Zhao W., Prijic S., Urban B.C., Tisza M.J., Zuo Y., Li L., Tan Z., Chen X., Mani S.A, & Chang J.T. (2016). Candidate Antimetastasis Drugs Suppress the Metastatic Capacity of Breast Cancer Cells by Reducing Membrane Fluidity. Cancer research, 76(7), 2037-2049.

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MCF-7 and ZR-75-1 Breast Cancer Cell Lines

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Human breast MCF-7 (ATCC, Rockville, MD, United States) tumor cells were grown in Phenol Red-free RPMI 1640 media and supplemented with 10% fetal bovine serum and antibiotics. Human breast ZR-75-1 tumor cells (ATCC, Rockville, MD, United States) were grown in Gibco’s DMEM medium containing glucose and glutamine and supplemented with 10% FBS and antibiotics. Both tumor cells were routinely used for 20–25 passages, after which the cells were discarded, and a new cell culture was started from the frozen stock. Three independent experiments of exponentially growing MCF-7 cells (70–75% confluency) were left untreated or treated with TPT (1 μM) for 1 and 24 h. ZR-75-1 cells were treated with TPT (1 μM) for 24 h. Cells were washed twice with PBS (pH 7.4) and total RNA was extracted with TRIzol (Ambion, Life Technologies, Grand Island, NY, United States) and RNeasy mini kit columns (Qiagen, Valencia, CA, United States).

Sinha B.K., Tokar E.J, & Bushel P.R. (2020). Elucidation of Mechanisms of Topotecan-Induced Cell Death in Human Breast MCF-7 Cancer Cells by Gene Expression Analysis. Frontiers in Genetics, 11, 775.

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Culturing Diverse Breast Cancer Cell Lines

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MCF10A, MCF7, BT474, T47D, ZR751, MDA-MB-157, MDA-MB-231, MDA-MB-361, MDA-MB-436, MDA-MB-453, MDA-MB-468, and HEK-293T were purchased from ATCC (Manassas, VA). MCF12A cells were a kind gift from Dr. Stefan Ambs (NIH, Bethesda). Immortalized human mammary epithelial cells MCF10A and MCF12A cells were cultured in DMEM/F12 media supplemented with 5% horse serum, 10 μg/ml human recombinant insulin (Sigma, Cat# 910077C), 20 ng/ml human EGF (PeproTech, Cat# AF100-15), 500 ng/ml hydrocortisone (Sigma, Cat# H0888g), 100 ng/ml cholera toxin (Sigma, Cat# C8052-5MG) and antibiotics. ATCC formulated Eagle’s Minimum Essential Medium supplemented with 10% FBS was used to culture MCF7 cells. All other cell lines were cultured in DMEM media supplemented with 10% FBS and antibiotics. All cell lines were maintained at 37°C in an incubator supplied with continuous 5% CO₂.

Malik N., Yan H., Yang H.H., Ayaz G., DuBois W., Tseng Y.C., Kim Y.I., Jiang S., Liu C., Lee M, & Huang J. (2021). CBFB cooperates with p53 to maintain TAp73 expression and suppress breast cancer. PLoS Genetics, 17(5), e1009553.

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Breast Cancer Cell Line Maintenance and Transfection

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The human breast cancer cell lines, MCF-7, T-47D, ZR-75–1, JIMT, SK-BR-3 and MDA-MB-231, were obtained from the ATCC and maintained in RPMI 1640 and Dulbecco's Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS) and 1% antibiotics (Invitrogen, San Diego, CA). Cells cultures were maintained at 37°C in an atmosphere of 5% CO₂, as previously described [34 (link)]. Transfection with CD44, CD44ICD, the truncated mutant CD44ICD, Sox2, and Oct4 expression vectors [35 (link)] as well as with CD44 siRNA was performed with Lipofectamine 2000 and Lipofectamine RNAiMAX reagents (Invitrogen), according to the reagent manufacturer's instruction. CD44 siRNA #1 (5′-UAUUCAAAUCGAUCUGCGCUU-3′) and CD44 siRNA #2 (5′-GACCAAUUACCAUAACUAUU-3′) were purchased from Genolution. Cells were harvested two days after transfection for use in the experiments.

Cho Y., Lee H.W., Kang H.G., Kim H.Y., Kim S.J, & Chun K.H. (2015). Cleaved CD44 intracellular domain supports activation of stemness factors and promotes tumorigenesis of breast cancer. Oncotarget, 6(11), 8709-8721.

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