Tissue cultures of primary breast tumors were performed as described (van der Kuip et al, 2006 (link)). Briefly, tissue slices (thickness, 200 μm) deriving from surgical specimens of 45 breast cancer patients who underwent a diagnostic Tru-cut procedure were challenged with vehicle (DMSO) or ATRA (0.1 μM) for 48 h in Mammary Epithelial Cell Growth Medium (Lonza, Allendale, NJ). At the end of the treatment, samples were fixed, paraffin-included, and dissected into 5-μm slices, which were subjected to immuno-histochemical staining with an antibody targeting the Ki67 proliferation-associated marker. The percentage of Ki67-positive tumor cells in the various samples was assessed in a quantitative manner by automatic image analysis, and the results are illustrated. Scoring of Ki67 was blinded as to treatment. Each value represents the mean ± SE of at least five separate fields for each experimental sample. The fresh primary tumor samples used for the short-term tissue slice cultures aimed at assessing ATRA sensitivity were supplied by Fondazione S. Maugeri, Pavia. All the procedures were approved by the internal ethical committee of the Fondazione S. Maugeri, and an informed consent for the donation of the sample was obtained from patients.
Mammary epithelial cell growth medium
Manufactured by Lonza
Sourced in Switzerland, United States
Mammary Epithelial Cell Growth Medium is a cell culture medium specifically formulated to support the growth and maintenance of primary human mammary epithelial cells in vitro. It provides the necessary nutrients and growth factors to facilitate the proliferation and differentiation of these cells.
Automatically generated - may contain errors
Lab products found in correlation
Mcf 10a, by Lonza (4 mentions)
Mcf 10a, by American Type Culture Collection (3 mentions)
Ga 1000, by Lonza (2 mentions)
Cholera toxin, by Merck Group (2 mentions)
Mda mb 231, by American Type Culture Collection (2 mentions)
Mcf 7, by American Type Culture Collection (2 mentions)
Mda mb 468, by American Type Culture Collection (2 mentions)
Bt474, by American Type Culture Collection (2 mentions)
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Trypsin edta, by Corning (1 mentions)
Megm bullet kit, by Lonza (1 mentions)
100mm ultra low attachment culture dishes, by Corning (1 mentions)
Dulbecco s modified eagle s medium dmem high glucose, by Merck Group (1 mentions)
Modified eagle s medium, by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions)
Cholera toxin, by Fujifilm (1 mentions)
Dulbecco s modified eagle s medium, by Fujifilm (1 mentions)
16 protocols using mammary epithelial cell growth medium
1
ATRA Sensitivity in Breast Cancer
2
Cell Culture Protocols for Breast Cancer Cell Lines
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All cell lines used for this study were purchased from the American Type Culture Collection ATCC and grown in a humidified incubator at 37°C with 5% CO2. MCF10A and HMEC cells were grown in Lonza’s Mammary Epithelial Cell Growth Medium (MEGM) supplemented with Lonza’s MEGM bullet kit containing, Bovine Pituitary Extract (BPE), human epidermal growth factor (hEGF), insulin, hydrocortisone and Gentamicin-Amphotericin (GA-1000). MDA-MB-468 and MDA-MB-231 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS, 1% penicillin/streptomycin and 1% amphotericin. HCC and 4T1 cells were maintained in RPMI supplemented with 10% FBS, 1% penicillin/streptomycin and 1% amphotericin and 4mM glutamine. All the supplements used were purchased from Corning Inc. PBS and trypsin-EDTA used for cell culture maintenance were also purchased from Corning Inc. and used as purchased.
3
Sphere formation of MCF7 cells
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MCF7 cells and their derivatives were seeded into 100-mm ultra-low attachment culture dishes (Corning) in Mammary Epithelial Cell Growth Medium (Lonza) at a density of at 2 × 104 cells per ml.
4
Arsenic Intoxication of Breast Cell Lines
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The double positive breast cancer cell line, MCF-7 was maintained in Minimum Essential Medium (MEM) with added 10% fetal bovine serum (FBS), 1% glutamine (Glu) and 1% Penicillin-Streptomycin (Pen-Strep). For intoxication, 1 μM of As2O3 were added in the complete culture media for 3 weeks or 6 weeks. The normal epithelial cell line, MCF-10A (CRL-10317™) was cultured in 50% Mammary Epithelial Cell Growth Medium from Lonza (Gampel, Switzerland) and 50% High Glucose Dulbecco’s Modified Eagle’s Medium (DMEM) from Sigma Aldrich (ST. Louis, USA), with added 2% FBS, 1% Glu and 1% Pen-Strep. For intoxication of MCF-10A cells, 1 μM of As2O3 was added in the complete culture media for 3 weeks or 6 weeks. The cells were maintained in a humidified incubator at 5% CO2 and 37 °C. The resulted intoxicated cell cultures were referred as: MCF-10A_As2O3_3w, MCF-10A_As2O3_6w, MCF-7_As2O3_3w and MCF-7_As2O3_6w.
5
Culturing Diverse Breast Cancer Cell Lines
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Normal human breast epithelial cell line MCF‐10A was cultured in Mammary Epithelial Cell Growth Medium (Lonza No. CC‐3150) with 100 ng/ml cholera toxin (2‐mg vials; Sigma No. C‐8052, Grand Island). Human breast cancer cell lines ZR‐75‐1, ZR‐75‐30, Hs578T were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco) containing 10% fetal bovine serum (FBS). Human breast cancer cell line MCF‐7 was cultured in modified Eagle's medium (Gibco) containing 10% FBS and 10 mg/l insulin. Human breast cancer cell line SK‐BR‐3 was cultured in McCoy’s 5a modified medium containing 10% FBS. Human breast cancer cell lines MDA‐MB‐453 and MDA‐MB‐231 were cultured in Leibovitz’s L‐15 containing 10% FBS. Human breast cancer cell line HCC1937 was cultured in Roswell Park Memorial Institute‐1640 (RPMI‐1640) medium containing 10% FBS. The cells were incubated at 37°C in a humidified incubator containing 5% CO2.
6
Establishing Breast Cancer Cell Lines
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The breast cancer cell lines (ZR-75-1, BT-474, MCF7, SK-BR-3, MDA-MB-231, and MDA-MB-468), and a non-tumorigenic epithelial cell line, MCF 10A, were purchased from American Type Culture Collection (ATCC; Manassas, VA, USA). The cancer cells were cultured in Dulbecco’s modified Eagle’s medium (WAKO Pure Chemical Industries, Ltd., Osaka, Japan) supplemented with 10% fetal bovine serum (Biosera, Nuaille, France), 100 U/mL penicillin, and 100 μg/mL streptomycin (Life Technologies, Carlsbad, CA, USA). MCF 10A cells were cultured in mammary epithelial cell growth medium (Lonza, Basel, Switzerland) supplemented with bovine pituitary extract, hydrocortisone, hEGF, insulin (Bullet Kit, Lonza) and 100 ng/mL cholera toxin (WAKO Pure Chemical Industries, Ltd.). All cell lines were cultured in a humidified atmosphere in 5% CO2 at 37 °C.
7
Evaluating Tumor Sphere Formation
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Tumor cells were cultured at 5 × 103, 1 × 104, and 5 × 104 cells/ml when using ex vivo cancer cells, or 1 × 103 cells/ml when using tumor lines, in 96-well ultra-low-attachment plates in mammary epithelial cell growth medium (Lonza) with 0.9% methylcellulose (Sigma Aldrich). After 3 to 4 weeks, >100 μm–diameter tumor spheres were microscopically counted in triplicate wells. For serial passaging, primary spheres were dissociated to single cells and replated in mammary epithelial cell growth medium. Cultures were monitored for secondary and tertiary sphere formation for up to 4 weeks. To block NKG2D-NKG2DL engagement, assays were in the presence of either a cocktail of antibodies (Abs) specific for MICA/B (6D4; BD Pharmingen) and ULBP1-6 [clones 170,818 (ULBP1), 165,903 (ULBP2,5,6), and 166,510 (ULBP3) from R&D Systems and clone 1H11 (ULBP4; 20 (link))], each at 10 μg/ml, or control isotype immunoglobulin (Ig), or the pan-NKG2DL-binding NKG2Dscd7 (10 μg/ml) or PBS control. Agonist anti-NKG2D Ab (clone 149,810) was from R&D Systems [26] (link).
8
Isolation and Culture of Breast Cancer Cells
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Breast tumor differentiated cells from two patients (#3,#4) and BTSCs (Breast tumor stem cells) were obtained as previously described [26 (link),45 (link)], and were used for the miR array. T47D (ER-positive) and MDA-MB-MB-231 (TNBC) differentiated cells were grown in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), 2 mM L-glutamine and 100 U/mL penicillin/streptomycin [46 (link)]. For mammosphere cultures, single cells were plated at a density of 1,000 cells/mL for T47D and 50,000/mL for MDA-MB-231. T47D and patients’ cells were grown in serum-free DMEM-F12 (Sigma, Milan, Italy), supplemented with B27 (Life technologies, Milan, Italy), 10 ng/mL EGF (Sigma), 20 ng/mL FGF (BD Biosciences, Milan, Italy) and 1X antibiotic-antimycotics (Life technologies). MDA-MB-231 stem cells were grown in Mammary Epithelial Cell Growth Medium (Lonza, Milan, Italy) supplemented with BPE, hEGF, insulin, hydrocortisone, GA-1000 (Lonza), B27 (Life technologies), and 20 ng/mL FGF (BD Biosciences, Milan, Italy). After 4–7 days, mammospheres, appearing as spheres of floating viable cells, were collected by gentle centrifugation (800 rpm) and dissociated with 0.25% trypsin for 5 min.
9
Cell Culture and EMT Induction
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Human embryonic kidney cell line 293FT (ATCC), colorectal carcinoma cell line HCT116 (ATCC), breast carcinoma cell line MDA-MB-231 derivative LM2 (from Dr. Yibin Kang at Princeton University), and PDX-derived HIM3 (provided by Dr. Helen Piwnica-Worms at MD Anderson) cell lines were cultured in DMEM supplemented with 10% FBS, l -glutamine, penicillin, and streptomycin. HMLE/Twist-ER cells (from Dr. Jing Yang at UCSD) were grown in Mammary Epithelial Cell Growth Medium (Lonza, USA). To induce EMT in HMLE/Twist-ER cells, a final concentration of 20 nM TAM was added to its culture medium, and cells were split every other day until the mesenchymal morphology was fully observed.
10
Curcumol Impacts Breast Cancer Cells
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The breast cancer cell lines MCF-7, MDA-MB-231 and the human normal breast epithelial cell MCF10A were purchased from Shanghai Cell Bank, Conservation Genetics, the Chinese Academy of Sciences(shanghai, China). The TAM-resistant cell lines (MCF-7 TAM-R) were provided with Dr ZY Wang. All cells were cultured in Dulbecco's modified Eagle's medium (Gibco, Grand Island, NY, USA) with 10% FBS (Gibco, Auckland, New Zealand), 100 U/mL penicillin and 100 μg/mL streptomycin at 37°C in a 5% CO2 incubator. In addition, MCF-7 TAM-R incubated in medium containing 1μM TAM to maintain resistance. MCF10A was cultured with Mammary Epithelial Cell Growth Medium (Lonza/Clonetics, Basel, Switzerland). The cells were pre-treated with different concentrations of curcumol (0, 12.5, 25, 50, 100 μg/mL). All cells are in the logarithmic growth phase for experiments.
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