Humec ready medium

Manufactured by Thermo Fisher Scientific

Sourced in United Kingdom

HuMEC Ready Medium is a cell culture medium designed for the growth and maintenance of human mammary epithelial cells. It provides the necessary nutrients and growth factors to support the in vitro cultivation of these cell types.

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HuMEC medium (15 citations) HuMEC (15 citations)

21 protocols using humec ready medium

Breast Cancer Cell Culture Protocol

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Human breast cancer MCF-7 (ATCC HTB-22) and BT-20 (ATCC HTB-19) cells were cultured in Dulbecco’s Modified Eagle Medium (Cleveland Clinic Cell Services Media Core, Cleveland, OH, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, Gaithersburg, MD, USA), 1% antibiotics-antimycotics (Gibco), 1% l-glutamine (Gibco), 1% nonessential amino acids (Gibco), and 1% sodium pyruvate (Gibco). Human nontumorigenic breast epithelial MCF-10A (ATCC CRL-10317) cells were cultured in HuMEC Ready Medium (Gibco). Cells were maintained in a humidified atmosphere with 5% CO₂ at 37°C. Cells were treated with drugs 24 hours after plating, incubated with drugs for an additional 72 hours, and collected for the different assays described below.^{36 (link),40 (link)}

Manouchehri J.M., Turner K.A, & Kalafatis M. (2018). TRAIL-Induced Apoptosis in TRAIL-Resistant Breast Carcinoma Through Quercetin Cotreatment. Breast Cancer : Basic and Clinical Research, 12, 1178223417749855.

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Cell Culture Maintenance Protocol

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Cell lines were all obtained from the American Type Culture Collection, or BioIVT. 293T, HeLa, MDA-MB-231, MDA-MB-468, HMEC, MCF7, MCF10A, SUM159, and BT474 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; GIBCO) supplemented with 10% FBS (Atlanta Biologicals) and 1% Pen/Strep (GIBCO). SUM149 cells were obtained from BioIVT and cultured in either F-12 medium (GIBCO) supplemented with 5% FBS (Atlanta Biologicals), 10mM HEPES pH7.5, 1μg/mL hydrocortisone, 5μg/mL insulin (GIBCO) and 1% Pen/Strep (GIBCO) or in HuMEC Ready Medium (GIBCO). All cells were incubated at 37°C and 5% CO₂.

Arceci A., Bonacci T., Wang X., Stewart K., Damrauer J.S., Hoadley K.A, & Emanuele M.J. (2019). FOXM1 Deubiquitination by USP21 Regulates Cell Cycle Progression and Paclitaxel Sensitivity in Basal-like Breast Cancer. Cell reports, 26(11), 3076-3086.e6.

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Cell Line Culture Conditions for Breast Cancer Research

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MDA-MB-231, MDA-MB-436, Hs578T, MCF-7, BT474, and 293T cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco 11965118) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (Pen Strep). T47D, HCC3153, HCC1187, HCC70 and MDA-MB-468 cells were cultured in 10% FBS, 1% Pen Strep RPMI 1640 (Gibco 11875093). Normal breast epithelial cells HMLE and MCF-10A were cultured in MEGM (Lonza CC-3151) containing SingleQuots Supplements (Lonza CC-4136). SUM149 cells were cultured in HuMEC Ready Medium (Gibco 12752–010). HCC3153 were obtained from the cell repository of the Hamon Center for Therapeutic Oncology Research, UT Southwestern Medical Center. HMLE was obtained from Dr. Wenjun Guo. All other cell lines were obtained from ATCC. Cells were used for experiments within 10–20 passages from thawing. All cells were authenticated via short tandem repeat testing. Mycoplasma detection was routinely performed to ensure cells were not infected with mycoplasma by using MycoAlert Detection kit (Lonza, LT07–218).

Liao C., Zhang Y., Fan C., Herring L.E., Liu J., Locasale J.W., Takada M., Zhou J., Zurlo G., Hu L., Simon J.M., Ptacek T.S., Andrianov V.G., Loza E., Peng Y., Yang H., Perou C.M, & Zhang Q. (2020). Identification of BBOX1 as a Therapeutic Target in Triple-Negative Breast Cancer. Cancer discovery, 10(11), 1706-1721.

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Cell Culture Maintenance Protocol

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Culturing and Treating Cancer Cell Lines

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MDA-MB-231, MDA-MB-436, Hs578T, BT474, and 293T cells were cultured in DMEM (Gibco 11965118) supplemented with 10% FBS and 1% penicillin–streptomycin. T47D, HCC3153, HCC70, and MDA-MB-468 cells were cultured in 10% FBS, 1% penicillin–streptomycin RPMI 1640 (Gibco, 11875093). Normal breast epithelial cells MCF10A were cultured in Mammary Epithelial Growth Medium (Lonza CC-3151) containing SingleQuots Supplements (Lonza CC-4136). SUM149 cells were cultured in HuMEC Ready Medium (Gibco, 12752–010). HCC3153 cells were obtained from the cell repository of the Hamon Center for Therapeutic Oncology Research, UT Southwestern Medical Center (Dallas, TX). Mycoplasma detection was routinely performed using the MycoAlert Detection Kit (Lonza, LT07–218). All cells were maintained at 37 °C in a humidified atmosphere containing 5% CO₂. For hypoxia treatment, cells were maintained for indicated time in the hypoxia chamber (Coy Laboratory Products). DMOG (D1070–1g, 1 μM) was from Frontier Scientific, DFO (D9533–1G, 200 mM) and FG4592 (D9533–1G, 200 mM) was from Sigma. For drug treatment, cells were treated with drugs for 12 hours.

Liu X., Wang J., Boyer J.A., Gong W., Zhao S., Xie L., Wu Q., Zhang C., Jain K., Guo Y., Rodriguez J., Li M., Uryu H., Liao C., Hu L., Zhou J., Shi X., Tsai Y.H., Yan Q., Luo W., Chen X., Strahl B.D., von Kriegsheim A., Zhang Q., Wang G.G., Baldwin A.S, & Zhang Q. (2022). Histone H3 proline 16 hydroxylation regulates mammalian gene expression. Nature genetics, 54(11), 1721-1735.

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Cultivation of Diverse Breast Cancer Cell Lines

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Breast cancer cell lines MDA-MB-231 and MCF-7 were purchased from ATCC and maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, UK) with 10% foetal calf serum (Gibco, UK). HCC38 cells were also purchased from ATCC and maintained in Roswell Park Memorial Institute (RPMI) medium (Gibco, UK) with 10% foetal calf serum (Gibco, UK). The 4 T1 breast cancer cell lines were kindly donated by Dr. Hideo Okada (University of California). The 4T1 cells were cultured in DMEM with 4 mM L-glutamine and charcoal-stripped bovine calf serum (10%). MCF10A cells were purchased from ATCC and maintained in HuMEC-ready medium (Thermo Fisher, UK) supplemented with HuMEC supplement kit (Thermo Fisher, UK). All cell lines were maintained in humid conditions at 37 °C and with 5% atmospheric CO_2. Cell lines were cultured in filtered tissue culture flasks (Fisher, UK) and passaged twice weekly when confluency was reached.

Flaherty R.L., Owen M., Fagan-Murphy A., Intabli H., Healy D., Patel A., Allen M.C., Patel B.A, & Flint M.S. (2017). Glucocorticoids induce production of reactive oxygen species/reactive nitrogen species and DNA damage through an iNOS mediated pathway in breast cancer. Breast Cancer Research : BCR, 19, 35.

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Cell Culture Protocols for Mammary Cell Lines

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V79 cells were purchased from Sigma-Aldrich (St. Louis, MO, USA). The cells were cultured in high glucose DMEM supplemented with 1% PS, 2 mM Glutamine and 10% FCS in a humidified incubator under 5% CO₂ and 37°C. Namru Mouse Mammary Gland (NMuMG) cells were kindly provided by Prof. R. Montesano (University of Geneva, Switzerland). The cells were grown in high glucose DMEM supplemented with 10% FCS and 1% PS. Michigan Cancer Foundation-10A (MCF-10A) cells were purchased from ATCC (Manassas, VA, USA). The cells were grown in DMEM/F12 Glutamax supplemented with 5% Horse serum, 1% PS, 10 ng/mL EGF, 5 µg/mL Insulin, and 1µM Dexamethasone. Primary Human Mammary Epithelial Cells (HMEC) cells were from Thermo Fisher Scientific (Waltham, MA, USA). The cells were received at passage 1, they were cultured in Humec Ready medium (Thermo Fisher Scientific; Waltham, MA, USA) and used at passage 2.

Tenan M.R., Nicolle A., Moralli D., Verbouwe E., Jankowska J.D., Durin M.A., Green C.M., Mandriota S.J, & Sappino A.P. (2021). Aluminum Enters Mammalian Cells and Destabilizes Chromosome Structure and Number. International Journal of Molecular Sciences, 22(17), 9515.

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Dissociation and culture of primary tumor cells

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Primary epithelial cells (from three distinct tumors (namely #1, #2, #3) from each mouse model or from three PDX implants) were obtained using the gentle MACS™ Dissociator and Mouse Tumor dissociation kit (Miltenyi Biotec, Bisley, Surrey, UK) following the manufacturer’s recommendations using the protocol for ‘Dissociation of Tough Tumors’ for mouse tumors and the protocol for ‘Dissociation of Soft and Medium Tumors’ for the PDX. To ensure efficient dissociation volumes of Enzyme D, Enzyme R and Enzyme A were scaled up according to the size of the tumor piece (100 μL, 50 μL and 12.5 μL respectively per each 0.5 cm³). The optional steps - the short spin for collection of the dissociated material at the bottom of the MACS tube and red blood cell lysis - were included in the procedure.
Mouse cells were cultured in complete growth medium in 2D adherent conditions for expansion or in 3D for functional studies. Cells up to passage 5 were used for all the experiments in this study. Freshly isolated human PDX cells were grown in HuMEC Ready Medium (Thermo Fisher Scientific) in Matrigel in 3D. Cultures were maintained at 37°C in a 5% CO₂/5%O₂ atmosphere in a Galaxy 170R incubator (New Brunswick, Eppendorf).

Tornillo G., Knowlson C., Kendrick H., Cooke J., Mirza H., Aurrekoetxea-Rodríguez I., Vivanco M.D., Buckley N.E., Grigoriadis A, & Smalley M.J. (2018). Dual Mechanisms of LYN Kinase Dysregulation Drive Aggressive Behavior in Breast Cancer Cells. Cell Reports, 25(13), 3674-3692.e10.

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Culturing and Characterizing Mammary Cell Lines

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The non-cancerous human mammary epithelial cell line MCF10A was obtained from American Type Culture Collection (ATCC; Manassas, VA) and cultured as reported [41 (link), 42 (link)]. MCF- 10DCIS.com (MCFDCIS) cells were purchased from Asterand (Detroit, MI) and cultured in MCF10A media. HuMECs were purchased from ATCC and cultured in HuMEC Ready Medium (Thermo Fisher Scientific; Waltham, MA). For EV collection, MCF10A and MCFDCIS cells were grown in media containing EV-depleted horse serum generated by spinning medium-diluted horse serum at 110,000 × g for 16 hr and discarding the EV pellet. The following antibodies were used for Western blot: CD9 (catalog # 13403; Cell Signaling Technology; Danvers, MA), CD63 (catalog # sc-59286; Santa Cruz Biotechnology; Santa Cruz, CA), TSG101 (catalog # MA1-23296; Thermo Fisher Scientific), and GM130 (catalog # 12480; Cell Signaling Technology). Rab27a/b knockdown cell lines were generated by clonal selection of cells transduced with lentivirus carrying MISSION pLKO.1-puro-RAB27A or -RAB27B shRNA (RAB27A: catalog # SHCLNG-NM_004580, TRC # TRCN0000380306; RAB27B: catalog # SHCLND-NM_004163, TRC # TRCN0000294016) or pLKO.1-puro empty vector control (catalog # SHC001) from Sigma-Aldrich (St. Louis, MO).

Chin A.R., Yan W., Cao M., Liu X, & Wang S.E. (2018). Polarized Secretion of Extracellular Vesicles by Mammary Epithelia. Journal of mammary gland biology and neoplasia, 23(3), 165-176.

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Characterizing Breast Cancer Cell Lines

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Nine breast cancer cell lines were studied: two ER+/PR+ (MCF‐7 and T47D), two HER2+ (HCC1954 and JIMT‐1) and five TNBC cell lines (MDA‐MB‐436, MDA‐MB‐231, MDA‐MB‐468, HCC1937 and Hs578T). In addition, non‐cancerous mammary epithelial cells (MCF‐10A and HuMEC) were also analysed. The cells were authenticated using Single Tandem Repeat analysis (STR), except HuMEC which was obtained from Thermo Fisher Scientific (MA, USA). All cells except MCF‐10A and HuMEC were cultured in high glucose Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum, 1x penicillin/streptomycin, 20 mM HEPES and 1x non‐essential amino acids (all from Thermo Fisher Scientific). DMEM/F12 medium supplemented with 5% (v/v) horse serum, 1x penicillin/streptomycin (all from Thermo Fisher Scientific), 20 ng/mL epidermal growth factor (PeproTech, NJ, USA), 10 µg/mL insulin, 100 ng/mL cholera toxin and 0.5 µg/mL hydrocortisone (all from Sigma–Aldrich, MO, USA) was used to culture MCF‐10A cells. HuMEC cells were maintained in HuMEC Ready Medium (Thermo Fisher Scientific). Cells were maintained at 37°C in 5% CO₂. Experiments for each cell line were performed in triplicate.

Dorado E., Doria M.L., Nagelkerke A., McKenzie J.S., Maneta‐Stavrakaki S., Whittaker T.E., Nicholson J.K., Coombes R.C., Stevens M.M, & Takats Z. (2024). Extracellular vesicles as a promising source of lipid biomarkers for breast cancer detection in blood plasma. Journal of Extracellular Vesicles, 13(3), e12419.

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