All human breast cancer cells, NMuMG cells, and 293T cells were obtained from American Type Culture Collection. HMLE cells were obtained from R. Weinberg (MIT, USA). MDAMB231, NMuMG, and 293T cells were maintained in DMEM supplemented with 10% FBS. MCF7 cells were maintained in DMEM supplemented with 10% FBS and 10 μg/ml insulin. BT474, HCC1143, BT549, and MDAMB468 cells were maintained in RPMI1640 supplemented with 10% FBS. SUM149 and SUM159 cells were maintained in Ham’s F12 supplemented with 5% FBS, 10 mM HEPES, 1 µg/ml Hydrocortisone, and 5 μg/ml Insulin. MCF10A and MCF12A cells were maintained in MEGM supplemented 100 ng/ml cholera toxin. HMLE cells were maintained in MEGM bullet kit (Lonza, no. CC-3150). All media were supplemented with 1% penicillin and streptomycin.
Megm bullet kit
Manufactured by Lonza
Sourced in Switzerland, United States, Italy
The MEGM bullet kit is a laboratory equipment product designed for specific research applications. It serves a core function related to cell culture and growth media preparation. Further details on the intended use of this product are not available.
Automatically generated - may contain errors
Lab products found in correlation
Mda mb 231, by American Type Culture Collection (14 mentions)
Mcf 10a, by American Type Culture Collection (13 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (13 mentions)
Mcf 7, by American Type Culture Collection (11 mentions)
Cholera toxin, by Merck Group (6 mentions)
Mcf 10a, by Lonza (6 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (6 mentions)
Mycoalert mycoplasma detection kit, by Lonza (5 mentions)
Mda mb 468, by American Type Culture Collection (5 mentions)
Zr 75 1, by American Type Culture Collection (5 mentions)
Mda mb 453, by American Type Culture Collection (5 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (5 mentions)
Skbr3, by American Type Culture Collection (5 mentions)
Bt474, by American Type Culture Collection (4 mentions)
Hs578t, by American Type Culture Collection (4 mentions)
Sum159, by American Type Culture Collection (4 mentions)
Bt549, by American Type Culture Collection (4 mentions)
Hydrocortisone, by Merck Group (3 mentions)
Glutamax, by Thermo Fisher Scientific (3 mentions)
Ovcar3, by American Type Culture Collection (3 mentions)
58 protocols using megm bullet kit
1
Comprehensive Breast Cancer Cell Culture
2
Cell Synchronization and FACS Analysis
3
Cytotoxic Evaluation of Cell Lines
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Human breast (MCF7), lung (A549) and prostate (PC3) cancer cell lines and non-tumorigenic breast (184B5) cell line were purchased from ATCC® (Virginia, USA). MCF7, PC3 and A549 cells were maintained in Roswell Park Memorial Institute 1640 medium (RPMI-1640, Biowest, France) supplemented with 10% fetal bovine serum (FBS). 184B5 cells were maintained in MEBM medium supplemented with MEGM bullet kit (Lonza, Basel, Switzerland) and 10% FBS. Cells were cultured in a humidified carbon dioxide (5%) incubator with temperature set at 37 °C.
The cytotoxic effect of extract and fractions was examined using the MTT assay as described in Mosmann (1983) (link) with some modifications. Cells with optimal density was seeded into a 96-well microplate and incubated overnight prior to treatment with various concentrations of samples for 72 h. At the end of the incubation time, 20 µL of MTT solution (5 mg/mL) was added and cells were incubated in the dark at 37 °C for 4 h. All the solution was removed by aspiration and 200 µL of DMSO was added to dissolve the purple formazan crystals. Absorbance readings were measured using a microplate reader at 570 nm. A plot showing percentage of cell viability against the tested sample concentration was used to determine the half-maximal inhibitory concentration (IC50).
The cytotoxic effect of extract and fractions was examined using the MTT assay as described in Mosmann (1983) (link) with some modifications. Cells with optimal density was seeded into a 96-well microplate and incubated overnight prior to treatment with various concentrations of samples for 72 h. At the end of the incubation time, 20 µL of MTT solution (5 mg/mL) was added and cells were incubated in the dark at 37 °C for 4 h. All the solution was removed by aspiration and 200 µL of DMSO was added to dissolve the purple formazan crystals. Absorbance readings were measured using a microplate reader at 570 nm. A plot showing percentage of cell viability against the tested sample concentration was used to determine the half-maximal inhibitory concentration (IC50).
4
Culturing Mouse Embryonic Stem Cells
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E14 mouse embryonic stem cells (mESCs) were cultured in high glucose DMEM media, supplemented with 15% ES grade fetal bovine serum (FBS; Biowest S182S‐500, Batch S10397S182), sodium pyruvate, gentamycin (Gibco 15710‐064), non‐essential amino acids (NEAA; Gibco 11140‐050), glutamax (Gibco 35050‐061), β‐mercaptoethanol (Gibco 21985023), and 1,000 U/ml LIF (ESGRO 1106). The mESCs were grown on 0.1% gelatin‐coated plastic and passaged every 2 days, with daily media changes, and for a maximum of 12 passages. mESCs were differentiated with 0.5 μM RA for 48 h, unless specified otherwise. HCT116 Myc‐Luc reporter cells were purchased from Amsbio, BPS Bioscience (#60520). MDA‐MB‐231 and HS578T breast cancer cell lines were gifts from Wai Leong TAM. The above were cultured in high glucose DMEM with sodium pyruvate, supplemented with 10% FBS (Biowest S181B‐500), 1% penicillin/streptomycin (Gibco 15140‐122), and 1% glutamax (Gibco 35050‐061). The HCT116 Myc‐Luc reporter line is additionally cultured under selection with 500 μg/ml geneticin. Human mammary epithelial cells (HMECs) 1–3 were a gift from Mathijs Voorhoeve. The HMECs were maintained in MEGM™ Mammary Epithelial Cell Growth Medium (Lonza MEGM BulletKit; CC‐3151 and CC‐4136).
5
Cell Line Maintenance and Genetic Manipulation
6
Establishment and Maintenance of Breast Cancer Cell Lines
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Cell lines (MCF-7, T47D, MDA-MB-453, BT474, ZR75-1, MDA-MB-231, MDA-MB-468, HER18, SUM149, SUM159, 293T and HS578T) were purchased from American Type Culture Collection (ATCC) and maintained in DMEM medium containing 10% fetal bovine serum. 76NF2V and hMLE MEC cells, generous gifts from Drs Vimla Band and Li Ma, respectively, were cultured in in MEGM Bullet kit (Lonza). Mycoplasma contamination was examined using MycoAlert mycoplasma detection kit (Lonza). Lentiviruses and retroviruses encoding short hairpin RNAs (Sigma) or complementary DNA were stably transduced into target cells and selected using puromycin (2 mg ml−1). For stable transfection, pcDNA3.1 mammalian expression plasmids (G418 for selection marker) were used. iCRT14, an inhibitor for β-catenin–TCF interaction was purchased from (Santa Cruz).
7
Epithelial Cell Culture and Protein Analysis
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Human primary mammalian epithelial cells (HMECs, obtained from Lonza) were cultured in MEGM BulletKit (CC-3150). MCF7 and MDA-MB-231 (MDA231, obtained from ATCC) were cultured in DMEM at 37°C supplemented with 10% FBS and antibiotics. Antibodies for β-actin (sc-47778), Ki-67 (sc-101861), SREBP1 (sc-13551), SREBP2 (sc-13552), and VDAC1 (sc-390996) were obtained from Santa Cruz Biotechnology. Antibodies for HKDC1 (ab228729) and PGC1β (ab176328) were obtained from Abcam. 3-nitrotyrosine (3-NT) was measured using the 3-Nitrotyrosine ELISA Kit (ab116691 from Abcam). The Coomassie Protein Assay Kit (Pierce Biotechnology) was used to measure the protein concentration. The siRNA for SREBP1, SREBP2, PGC1β, and negative control (#AM4636) were purchased from Ambion. The Lipofectamine™ Reagent (Invitrogen) was used for DNA transfection (5 (link)).
8
Breast Cancer Cell Line Culture and PIWIL Protein Analysis
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MCF-7 (ATCC HTB-22), SKBR3 (ATCC HTB-30) and ZR-75.1 (ATCC CRL-1500) cell lines were maintained in Dulbecco's modified Eagle medium (DMEM; Sigma-Aldrich, Milan, Italy) supplemented with 10% fetal bovine serum (FBS) (HyClone, Cramlington, UK) and antibiotics: 100U/ml penicillin, 100mg/ml streptomycin, 250ng/ml Amfotericin-B (exponential growing condition). MCF10A (ATCC CRL-10317) mammary epithelial cells were maintained in MEGM Bullet Kit (Lonza, Milan, Italy) supplemented with 100 ng/ml cholera toxin (Sigma-Aldrich, Milan, Italy). Steroid deprivation (starvation) was performed by culturing cells in phenol red-free DMEM and 5% Dextran-Coated Charcoal stripped serum (DCC-FBS) for 5 days, as described earlier [24 (link)]. PIWIL proteins expression was analyzed by sodium dodecyl sulphate (SDS) acrylamide gel electrophoresis and immunoblotting of total protein extracts, using rabbit anti-PIWIL1 (ab85125, Abcam, Cambrige, UK), rabbit anti-PIWIL2 (ab26408, Abcam), mouse anti-PIWIL3 (ab77088, Abcam), rabbit anti-PIWIL4 (ab111714, Abcam) and mouse anti-β-actin (A1978, Sigma Aldrich).
9
Cell Line Culturing Protocol for TNBC Research
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Five human TNBC cell lines, MDA-MB-231, BT-549, MDA-MB-468, MDA-MB-453 and SUM-159, as well as normal breast epithelial cell line MCF-10A were purchased from ATCC. 293 T and 293 cell lines were conserved in our laboratory. MDA-MB-231, MDA-MB-468, MDA-MB-453, SUM-159, 293 T and 293 cells were cultured in DMEM medium (Gibco, Carlsbad, CA, USA), BT-549 cells were grown in RPMI 1640 medium (Gibco, Carlsbad, CA, USA). MCF-10A cells were maintained in mammary epithelial cell growth medium (MEGM) BulletKit (Lonza, Basel, Switzerland). The cell media contained 10% fetal bovine serum (FBS, HyClone, Invitrogen), 100 U/ml penicillin and 100 mg/ml, cells were maintained in a humidified incubator at 37 °C with 5% CO2.
10
Genomic Characterization of Sarcoma and Ovarian Carcinoma Cells
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Patient‐derived sarcoma cells were genomically characterized by the FoundationOne®HEME assay and Infinium Human Methylation EPIC 850 k array (Illumina) targeted gene sequencing panel and maintained in DMEM (Gibco) with 10% fetal bovine serum (Gibco). Ovarian carcinoma cells UWB1.289 (ATCC, CRL‐2945, RRID:CVCL_B079) were genomically characterized by the FoundationOne®CDX assay targeted gene sequencing panel, which confirmed a pathogenic frameshift mutation in BRCA1 (2475delC). Cells were maintained in 1:1 RPMI‐1640 medium (Gibco) and Mammary Epithelial Cell Growth Medium (MEGM Bullet kit, Lonza, CC‐3150). All cells were passaged when sub‐confluent conditions were reached in cell culture‐treated flasks, incubated at 37°C in a humidified atmosphere with 5% CO2 and routinely tested for Mycoplasma.
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