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7 protocols using mcf 7

1

Breast Cancer Cell Line Cultivation

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The cancer cell lines used in this study were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA). They consisted of MDA-MB-231, MDA-MB-436 (ER-, PR- and HER2-), and MCF-7 (ER+, PR+ and HER2+) breast cancer cells, and the MCF-10A cell line (nontumorigenic epithelial cell line) was included to represent normal cells.
MDA-MB-231 and MCF-7 were grown in high-glucose DMEM supplemented with 5% fetal bovine serum (FBS, Biowest, Miami, FL, USA), while MDA-MB-436 cells were grown in DMEM/F12 supplemented with 10% FBS (Biowest, Miami, FL, USA).
MCF-10A cells were cultured in DMEM/F-12 supplemented with 5% horse serum (Biowest, Miami, FL, USA), 20 ng/mL epidermal growth factor (Upstate Biotechnology Incorporated, Lake Placid, NY, USA), 10 µg/mL insulin (Biofluids, Rockville, MD, USA), and 500 ng/mL hydrocortisone.
All media (Biowest, Miami, FL, USA) were also supplemented with 2 mM glutamine, 100 U/mL penicillin, and 100 mg/mL streptomycin. The cells were maintained in a humidified incubator at 37 °C with 5% CO2.
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2

Breast Cancer Cell Line Cultivation Protocol

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Gefitinib, lapatinib, terazosin, alfuzosin, prazosin, irinotecan, and quinacrine were purchased from Sigma Chemical (St. Louis, MO, USA). The breast cancer cell lines used in this study, MCF-7 and MDA-MB-231, were obtained from the American Type Tissue Culture Collection (ATCC), Rockville, MD, USA. MCF-7 and MDA-MB-231 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) high glucose with phenol red, and the culture medium was supplemented with 10% fetal bovine serum (FBS) (BioWest, Riverside, MO, USA) and 1% penicillin/streptomycin as an antibiotic. The cells were incubated in culture flasks (75 cm3) at 37 °C in a humidified atmosphere of 5% CO2 and 95% air, all of which were carried out under sterile conditions using a laminar flow hood.
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3

Culturing Epithelial and Cancer Cell Lines

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The normal human mammary epithelial cells (MCF-10A), human breast cancer cells (MCF-7), human ovarian cancer cells (A278), and human ovarian fibroblasts (HOF) healthy cell lines were purchased from American Type Culture Collection (ATCC). MCF-7 was grown in the Dulbecco’s Modified Eagle’s Medium-DMEM (BioWest) containing 1% mixture of penicillin and streptomycin (BioWest), 1% of L-glutamine (BioWest) and 10% fetal bovine serum (FBS, Gibco). To cultivate MCF-10A the Dulbecco's Modified Eagle Medium, nutrient mixture F-12 was used. The medium was supplemented with 5% horse serum (Sigma-Aldrich), 10 ng·mL−1 epithelial growth factor (Sigma-Aldrich), 5 μg·mL−1 hydrocortisone (Sigma-Aldrich) and 10 μg·mL−1 human insulin (Sigma-Aldrich). All cell cultures were mycoplasma free. To cultivate the A2780 cell lines the RPMI 1640 (BioWest) containing 1% mixture of penicillin and streptomycin (BioWest), 10% fetal bovine serum (FBS, Gibco) and 1% of L-glutamine (BioWest) was used. The HOF cells were cultivated by fibroblast medium that consisted of 500 ml of basal medium, 10 ml of fetal bovine serum (FBS), 5 ml of fibroblasts growth supplement (FGS), and 5 ml of antibiotic solution (P/S).
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4

Cell Culture Maintenance Protocol

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All cell lines used in this study were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). A549, NCI-H1975, NCI-H226, HCC827, MCF-7, and BEAS-2B, an immortalized human bronchial epithelial cell line, cells were maintained in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS, BioWest, Riverside, MO, USA) and 1% penicillin/streptomycin (Gibco, Waltham, MA, USA). BZR, viral H-Ras (H-RasG12V) transformed BEAS-2B cells, LLC and MDA-MB-231 cells were cultured in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin. Human umbilical vein endothelial cells (HUVECs) were purchased from ATCC and were cultured in M199 medium containing 20% heat-inactivated FBS, 1% penicillin/streptomycin, 15 μg/ml endothelial cell growth supplement (ECGS, Corning, NY, USA) and 5 units/ml of heparin (Sigma-Aldrich, St Louis, MO, USA). All cells were maintained in a humidified 5% CO2 atmosphere at 37 °C.
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5

Culturing Diverse Breast Cell Lines

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The human mammary epithelial cell line MCF10A and human breast cancer cell lines MCF7, T-47D, SK-BR-3, MDA-MB-231, MDA-MB-468 and BT-549 were bought from ATCC (Rockville, MD, USA). MCF10A was cultured in MEGM with100 ng/ml cholera toxin (ATCC, USA). MCF7, SK-BR-3 and MDA-MB-468 were cultured in DMEM with 10% fetal bovine serum (FBS, Biowest). T-47D and BT-549 were cultured in RPMI 1640 with 10% FBS. MDA-MB-231 was cultured in L-15 with 10% FBS. All cell lines were cultured at 37 °C in a humidified atmosphere containing 5% CO2.
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6

Culturing Luminal A Breast Cancer Cells

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T47D and MCF-7 breast cancer cell lines of the Luminal A subtype (American Type Culture Collection (ATCC), Manassas, Virginia) were used. The MCF-7 cell line was cultured in DMEM (Biowest, France) supplemented with 10% FBS, 1% P/S, and 0,01 mg/mL insulin. The T47D line was cultured in RPMI-1640 (Thermo Fisher Scientific, France) with 10% FBS and 1% P/S. Cells were passaged with 0.25% trypsin-EDTA (Merck Millipore Corporation, Germany) when they reached 80–90% confluency.
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7

Cell Culture Protocols for Human Cell Lines

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Experiments were carried out on four human cell lines: keratinocyte—HaCaT (purchased from ThermoFisher (Waltham, MA, USA)), malignant melanoma—A375 (purchased from ATCC (Manassas, VA, USA)), breast adenocarcinoma—MCF-7 (purchased from ATCC (Manassas, VA, USA)), and lung carcinoma—A549 (purchased from ATCC (Manassas, VA, USA)). The culture medium for HaCaT, A375, and MCF-7 cells was based on Dulbecco’s modified Eagle’s medium (DMEM, Biowest (Nuaille, France)), supplemented with 10% of fetal bovine serum (FBS, Biowest (Nuaille, France)), 1% 25 mM l-glutamine (Biowest (Nuaille, France)), 1% 100 mM penicillin, and streptomycin (Biowest (Nuaille, France)). The culture medium for A549 differed only in the base; it consisted of minimum essential medium (MEM, Biowest (Nuaille, France)) with the same additives as before. The cells were sub-cultured every two days using Tryple Express (Gibco (Waltham, MA, USA)) solution to detachment. Cells were kept at 37 °C and 5% CO2 in a humidified atmosphere.
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