ER- human breast non-tumorigenic epithelial cell lines (MCF-10A, MCF-10F and MCF-10-2A), ER+ human breast cancer cell lines (MCF-7 and T-47D) and ER- human breast cancer cell lines MDA-MB231 and SKBR-3) were from ATCC (Manassas, Virginia, USA). Tamoxifen was from Sigma-Aldrich. Cell culture medium (DMEM/F12), OPTI-MEM, Lipofectamine 2000 and TRIzol reagent were from Life Technologies (San Diego, CA, USA). Antibodies against β-actin and TFIIIC63 and c-Jun siRNA (Catalog No. SC-29224) were obtained from Santa Cruz Biotech (Santa Cruz, CA, USA). Mismatch RNA was described previously [24 (link)]. Histone H3 antibody were from Cell Signaling (Danvers, MA, USA). Brf1 antibody was from Bethyl laboratories Inc (Montgomery, TX, USA). The sequences of primers were described in (Supplements) [8 (link), 30 (link)].
Mcf 10 2a
Manufactured by American Type Culture Collection
Sourced in United States
The MCF-10-2A is a cell line derived from human mammary epithelial cells. It is a well-characterized model used for studies related to breast cancer and cell biology.
Automatically generated - may contain errors
Lab products found in correlation
Mda mb 231, by American Type Culture Collection (3 mentions)
Mcf 7, by American Type Culture Collection (3 mentions)
Cholera toxin, by Merck Group (2 mentions)
Epidermal growth factor (egf), by Merck Group (2 mentions)
Insulin, by Merck Group (2 mentions)
Hydrocortisone, by Merck Group (2 mentions)
Skbr3, by American Type Culture Collection (1 mentions)
Mcf 10a, by American Type Culture Collection (1 mentions)
Mcf 10f, by American Type Culture Collection (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Histone h3 antibody, by Cell Signaling Technology (1 mentions)
Tamoxifen, by Merck Group (1 mentions)
Brf1 antibody, by Fortis Life Sciences (1 mentions)
Epidermal growth factor (egf), by American Type Culture Collection (1 mentions)
Hydrocortisone, by American Type Culture Collection (1 mentions)
Cholera toxin, by American Type Culture Collection (1 mentions)
Insulin, by American Type Culture Collection (1 mentions)
Penicillin streptomycin, by Merck Group (1 mentions)
Penicillin streptomycin, by Harvard Bioscience (1 mentions)
7 protocols using mcf 10 2a
1
Breast Cancer Cell Lines Characterization
2
SENP7S Knockdown in Breast Cancer Cells
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MCF10-2A and MCF7 were purchased from ATCC (Manassas, VA, USA). MCF10-2A cells were grown in DMEM:F12 media including supplements EGFR, cholera toxin, insulin, hydrocortisone, 5% horse serum, and 0.5% penicillin/streptomycin. MCF7 cells were cultured in DMEM media with 10% FBS and 1% penicillin/streptomycin.
The SENP7S-siRNA oligo was integrated into a puromycin-selection shRNA vector (Origene) to generate SENP7S-targeting shRNA (shSENP7S). Subsequently, stable shSENP7S MCF10-2A clones were selected based puromycin resistance; a similar approach was utilized with a control non-targeting shRNA vector (shNT).
The SENP7S-siRNA oligo was integrated into a puromycin-selection shRNA vector (Origene) to generate SENP7S-targeting shRNA (shSENP7S). Subsequently, stable shSENP7S MCF10-2A clones were selected based puromycin resistance; a similar approach was utilized with a control non-targeting shRNA vector (shNT).
3
Culturing Breast Cancer Cell Lines
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Human breast cancer cell lines T-47D (HTB-113; ATCC), Hs 578T (HTB-126; ATCC) and MDA-MB-231 (HTB-26; ATCC) were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. MCF-10-2A (CRL-10781; ATCC) cells were grown in DMEM-F12 medium supplemented with 5% horse serum, 1% penicillin/streptomycin, 20 ng/ml epidermal growth factor (EGF), 100 ng/ml cholera toxin, 0.01 mg/ml insulin and 500 ng/ml hydrocortisone. All cell lines were maintained at 37°C in a humidified atmosphere with 5% CO2. For all experiments, cells were seeded in 6-well at a concentration of 1.5×105 cells/ml for 24 h experiments and 1×105 cells/ml for 48 h experiments, with the exception of the ECAR experiments in which cells were seeded in XF 24-well plates at a concentration of 1×104 cells/well. All medium constituents were acquired from Biochrom with the exception of EGF, cholera toxin, hydrocortisone and insulin that were purchased from Sigma-Aldrich.
4
Cell Culture of Breast Cancer Lines
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The human breast cancer cell line SK-BR-3 (ATCC HTB-30; provided by Dr. Ana Preto from the Centre of Molecular and Environmental Biology [CBMA-University of Minho]) and the BT-474 (ATCC HTB-20; provided by Dr. Amelia Silva from the University of Trás-os-Montes and Alto Douro) were grown in Dulbecco's Modified Eagle's Medium (DMEM, Biochrom) and DMEM:HAMs F-12, respectively, supplemented with 10% (v/v) fetal bovine serum (FBS, Biochrom) and 1% (v/v) of penicillin-streptomycin (Biochrom). The human non-tumorigenic mammalian cell line MCF-10-2A (ATCC CRL-10781) obtained from ATCC was routinely cultivated in a 1:1 solution of DMEM:HAMs F-12 medium supplemented with 5% horse serum (Merck Millipore), 20 ng/ ml epidermal growth factor (Merck Millipore), 100 ng/ml cholera toxin (Sigma-Aldrich), 0.01 mg/ml insulin (Sigma-Aldrich), 500 ng/ml hydrocortisone, 95% (Sigma-Aldrich) and 1% penicillin-streptomycin. Cells were maintained at 37ºC with 5% CO 2 . Sub-culturing was performed when the cell culture reached 80% of confluence. The cells were washed using phosphate-buffered saline 1× pH 7.4 (PBS 1×:137 mM NaCl [Nzytech], 2.7 mM KCl [ChemLab], 10 mM Na 2 HPO 4 [Scharlau] and 2 mM KH 2 PO 4 [Panreac] ) and detached using Trypsin/EDTA solution [Biochrom].
5
Culturing Breast Cancer Cell Lines
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MDA-MB-231, MCF-7 and MCF-10-2A cell lines were obtained from the American Type Culture Collection (ATCC). MDA-MB-231 were maintained in Leibovitz’s L15 medium (Life Technologies) supplemented with 10% FBS and antibiotic-antimytotic. MCF-7 cells were maintained in Eagle’s Minimum Essential Medium (EMEM, Life Technologies) supplemented with 10% FBS, antibiotic-antimytotic and 0.01 mg/ml bovine insulin (Life Technologies). MCF-10-2A cells were maintained in Dulbeccos modified Eagle medium (DMEM)/Ham’s F12 medium (Life Technologies) with 10% horse serum, 0.01 mg/ml bovine insulin, 20 ng/ml epidermal growth factor, 100 ng/ml Cholera toxin, 500 ng/ml hydrocortisone and antibiotic-antimytotic. Cells were incubated at 37 °C, MCF-7 and MCF-10-2A cells in the presence of 5% CO2.
6
Culturing MDA-MB231 and MCF102A Cells
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MDA-MB231 and MCF102A cell lines were obtained from the American Type Culture Collection (ATCC) (LGC Standard, Teddington, UK). The MDA-MB231 cell line was grown in Dulbecco’s modified Eagle’s medium (DMEM) containing 4 mM of L-glutamine, supplemented with 10% fetal bovine serum. The MCF102A cell line was maintained in DMEM/F-12 containing 5% horse serum, 2.5 mM L-glutamine, 20 ng/mL EGF, 100 ng/mL cholera toxin, 0.01 mg/mL insulin, and 500 ng/mL hydrocortisone. All media were supplemented with 100 U/mL penicillin and 100 µg/mL streptomycin, and cells were incubated at 37 °C in a 5% CO2 incubator. All reagents for media were purchased from Life Technologies, Waltham, MA, USA.
7
Inhibition of PLK4 in Cancer Cell Lines
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We used breast cancer (HCC1569, HCC1954, BT549, MDA-MB-415, HCC202, MCF7, T47D, CAL51), ovarian cancer (KOC-7C, SKOV3, HEYC2, COLO704, RMG-1, KK, ES-2, EFO21, MCAS, A2780, OV167, PEA2, TOV-21G, OV90, OV207, Kuramochi, OVISE, OVMANA, PEO6, CaOv-3, OV177, OVCAR3, OVCAR5, OVSAHO, OVTOKO, PEO14), colorectal cancer (HCT-116, HCT-15, DLD-1, COLO 205, COLO320DM, COLO320HSR, NCI-H747, NCI-H716, COLO201, LS1034, SNU-C2B, SNU-C1, LS513, LS411N, ATR-FLOX, LS174t, WiDr, RKO, SK-CO1, HCT-8, LS-180, HT-29, C2Bbe1, SW480), and immortalized breast epithelial (MCF-10–2A, MCF-12A) cell lines from the American Type Culture Collection (ATCC) or the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) for our experiments grown as described elsewhere (42 (link)–44 (link, link)). Cultured cells were synchronized as needed by using single or double thymidine blockade. For experiments with CFI-400945, the PLK4 inhibitor was added to medium at 10 nM, 50 nM, 100 nM, or 500 nM concentrations and compared with results with DMSO controls.
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