Experiments were performed on the MCF-10A cell line purchased from ATCC (Manassas, VA, USA). Cells were cultured in 2D in DMEM/F12 (Life Technologies, Carlsbad, CA, USA) containing 5% horse serum (New Zealand origin, Life Technologies, Carlsbad, CA, USA), 20 ng/mL EGF (Sigma-Aldrich, St. Louis, MO, USA), 0.5 μg/mL hydrocortisone, 100 ng/mL cholera toxin, 10 μg/mL insulin, 100 U/mL penicillin/streptomycin (all Sigma-Aldrich). Acini from MCF-10A were cultured as previously described [30 (link)]. In brief, single cells were seeded onto a Geltrex (ThermoFisher Scientific, Waltham, MA, USA) bed and cultivated for nine days in EGF-supplemented assay medium (DMEM/F12 Glutamax, 2% horse serum, 5 ng/mL EGF (Sigma-Aldrich, St. Louis, MO, USA), 0.5 μg/mL hydrocortisone, 1 ng/mL cholera toxin, 10 μg/mL insulin, 100 U/mL penicillin/streptomycin), changing the medium every three days. At day nine, EGF was eliminated from the assay medium for further cultivation, changing the medium every three days [30 (link)], while acini cultivation did not exceed 21 days.
Mcf 10a cell line
Manufactured by American Type Culture Collection
Sourced in United States
The MCF-10A cell line is a human breast epithelial cell line derived from a patient with fibrocystic breast changes. The cells are nontumorigenic and can be used to study normal breast epithelial cell biology and as a control for breast cancer research.
Automatically generated - may contain errors
Lab products found in correlation
Mda mb 231, by American Type Culture Collection (12 mentions)
Mcf 7, by American Type Culture Collection (9 mentions)
Cholera toxin, by Merck Group (7 mentions)
Insulin, by Merck Group (7 mentions)
Mda mb 468, by American Type Culture Collection (6 mentions)
Horse serum, by Thermo Fisher Scientific (6 mentions)
Epidermal growth factor (egf), by Merck Group (6 mentions)
Hydrocortisone, by Merck Group (6 mentions)
Dmem f12, by Thermo Fisher Scientific (5 mentions)
Mcf 10a, by Thermo Fisher Scientific (5 mentions)
Rpmi 1640, by Thermo Fisher Scientific (4 mentions)
Bt474, by American Type Culture Collection (4 mentions)
Mcf 10a, by Merck Group (4 mentions)
Skbr3, by American Type Culture Collection (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Dulbecco modified eagle medium (dmem), by Corning (3 mentions)
Dmem f12, by Merck Group (3 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (3 mentions)
Mda mb 435, by American Type Culture Collection (3 mentions)
Penicillin streptomycin, by Merck Group (2 mentions)
45 protocols using mcf 10a cell line
1
MCF-10A 3D Culture Protocol
2
Breast Cancer Cell Line Characterization
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The MCF-7 cell line, a noninvasive estrogen receptor positive (ER+) and MDA-MB-231 cell line ER negative were purchased from the American Type Culture Collection (ATCC, Manassas, VA). MCF-10A cell line, a non-tumorigenic epithelial cell line was also purchased from ATCC. 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium (MTT), Dulbecco's Modified Eagle's Medium (DMEM) with high glucose and FluoroBrite™ phenol red-free DMEM, (MCF-7) were purchased from Thermo Fisher Scientific (Rockford, IL) The RPMI 1640 medium (MDA-MB-231 cells) was obtained from Life Technologies Company (Carlsbad, CA). The basal medium MEBM and the additive MEGM (MCF-10A cells) were obtained from Lonzal/Clonetics Corporation (Lonza, Walkersville, MD). Paclitaxel was purchased from Sellck-Chemon, (Houston, TX). Cisplatin was purchased from EMD/Millipore (Billerica, MA). Annexin V-FITC/PI Apopto and PI Cell Cycle Kits were purchased from Nexcelom Bioscience (Lawrence, MA). The CD63 Rabbit polyclonal and Alix goat polyclonal antibodies were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). The SMRwt and SMRmut peptide sequences are described in [25 (link)]. For these experiments, peptides were mofdified by addition of polyethylene glycol (PEG) and a Clusterin-binding peptide. The PEG-SMRwt-Clu PEG-SMRwt and PEG-SMRmut HIV-1 Nef peptides were synthesized by the InnoPep Company (San Diego, CA).
3
TGF-β1 Induced EMT in MCF10A
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The MCF10A cell line was obtained from ATCC (Manassas, VA, USA) and cultured with MEGM (Lonza/Clonetics Corporation, Guangzhou, China) without GA-1000. Cholera toxin (B8326, ApexBio Technology, TX, USA) was added to a final concentration of 100 ng/mL. MCF10A cells were treated for 3 days with TGF-β1 (5 or 10 ng/mL, PHG9214, Life Technologies, Beijing, China). MDA MB-231 cell line was obtained from ATCC and cultured with RPMI 1640 (Gibco, LifeTechnologies, Beijing, China) with 10% fetal bovine serum.
4
Breast Cancer Cell Lines Characterization and Treatment
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MDA-MB-231 (hereafter called MDA) (ER−, Pr−, Her2−), MCF7 (ER+, PR+, Her2−), SKBR3 (ER−, Pr−, Her2+), BT-474 (ER+, Pr+/−, /Her2+) cell lines were from the ICLC (Interlab Cell Line Collection, Genoa, Italy). HeLa cell line was from ATCC. All cell lines were maintained in RPMI-1640 or DMEM supplemented with L-glutamine, antibiotics and 10% heat-inactivated fetal bovine serum (FBS, all from Sigma-Aldrich) according to the ICLC indications. MCF 10A cell line was from ATCC. Cells maintained in Dulbecco's modified Eagle's medium (DMEM)/F-12 medium containing 5% horse serum, hydrocortisone (0.5 μg/ml), insulin (10 μg/ml), EGF (20 ng/ml), cholera toxin (100 ng/ml), penicillin 100 units/ml) and streptomycin (100 lg/ml) at 37°C in a 5% CO2 humidified atmosphere. All cell lines from ICLC or ATCC were characterized by DNA (SRT) profiling. Cells were immediately expanded and frozen such that they could be revived every 3 to 4 months.
Cells were cultured in a humidified incubator in an atmosphere of 5% CO2 at 37°C. Before any experiment, cells were detached by mild trypsinization, washed, plated in complete medium and allowed to recover for 24 h.
Cells were treated with DHA (Sellekchem) or BI 2536 (Selleckchem) dissolved in DMSO and diluted in culture medium immediately before use. Control media contained the same amount of DMSO-vehicle (<0.1%).
Cells were cultured in a humidified incubator in an atmosphere of 5% CO2 at 37°C. Before any experiment, cells were detached by mild trypsinization, washed, plated in complete medium and allowed to recover for 24 h.
Cells were treated with DHA (Sellekchem) or BI 2536 (Selleckchem) dissolved in DMSO and diluted in culture medium immediately before use. Control media contained the same amount of DMSO-vehicle (<0.1%).
5
Mammalian Cell Culture Protocols
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HEK293T cell line (ATCC Cat# CRL-3216, RRID:CVCL_0063): maintained in DMEM (Corning®) supplemented with 10% fetal bovine serum (Corning®), 1% Penicillin-Streptomycin (Corning®), and prophylactic Plasmocin™ (InvivoGen) at 37°C with 5% CO2 and ≥ 80% relative humidity.
Expi293F cell line (Gibco™ Cat# A14527, RRID:CVCL_D615): maintained in Expi293™ Expression Medium (Gibco™) at 37°C with 8% CO2 and ≥ 80% relative humidity and 125 rpm shaking.
MCF-10A cell line (ATCC Cat# CRL-10317, RRID:CVCL_0598): maintained in DMEM/F12 (Corning®) supplemented with 5% horse serum (Invitrogen), 20 ng/mL EGF, 0.5 μg/mL Hydrocortisone, 100 ng/mL Cholera toxin, 10 μg/mL Insulin, 1% Penicillin-Streptomycin (Corning®), and prophylactic Plasmocin™ (InvivoGen) at 37°C with 5% CO2 and ≥ 80% relative humidity.
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HEK293T cell line (ATCC Cat# CRL-3216, RRID:CVCL_0063): maintained in DMEM (Corning®) supplemented with 10% fetal bovine serum (Corning®), 1% Penicillin-Streptomycin (Corning®), and prophylactic Plasmocin™ (InvivoGen) at 37°C with 5% CO2 and ≥ 80% relative humidity.
Expi293F cell line (Gibco™ Cat# A14527, RRID:CVCL_D615): maintained in Expi293™ Expression Medium (Gibco™) at 37°C with 8% CO2 and ≥ 80% relative humidity and 125 rpm shaking.
MCF-10A cell line (ATCC Cat# CRL-10317, RRID:CVCL_0598): maintained in DMEM/F12 (Corning®) supplemented with 5% horse serum (Invitrogen), 20 ng/mL EGF, 0.5 μg/mL Hydrocortisone, 100 ng/mL Cholera toxin, 10 μg/mL Insulin, 1% Penicillin-Streptomycin (Corning®), and prophylactic Plasmocin™ (InvivoGen) at 37°C with 5% CO2 and ≥ 80% relative humidity.
6
Culturing Diverse Breast Cancer Cell Lines
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MCF10A cell line was purchased from ATCC (Manassas, VA, USA). MCFNeoT, MCFT1, and MCFCA1 cells were obtained from the Barbara Ann Karmanos Cancer Institute (Detroit, MI). The four cell lines were maintained as monolayers in Dulbecco’s Modified Eagle’s Medium-F12 (DMEM/F12) (Gibco, 11320033) supplemented with 5% horse serum (Gibco, 16050122), 1% penicillin/streptomycin (Lonza, 17-602E), 0.5 μg/ml hydrocortisone (StemCell, 37150), 100 ng/ml cholera toxin (Sigma, C-8052), 10 μg/ml insulin (Gibco, 1285014), and 20 ng/ml recombinant human EGF (Invitrogen, PHG0311). THP-1 monocyte cell line was purchased from ATCC (TIB-202, Manassas, VI) and TNF-reporter cell line (THP1-B5) was a gift from Dr. Ian Fraser (NIH/NIAID). Cells were cultured in RPMI-1640 (Life Technologies, Carslbad, CA) supplemented with 10% FBS, 1% penicillin/streptomycin and 1% Sodium Pyruvate. MCF cells were trypsinized off the plates. All cell lines were regularly tested for Mycoplasma contamination by PCR.
7
Establishing TNBC Cell Line Cultures
8
Knockdown of LINC01016 in Breast Cancer
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The MDA-MB-231, MDA-MB-468, T47D, MDA-MB-453 human BC cell lines and MCF-10A cell line were purchased from ATCC. Three different Antisense oligonucleotides (ASOs) specific for LINC01016 (si-LINC01016-1, si-LINC01016-2, si-LINC01016-3 and si-LINC01016-4) were obtained from RiboBio (Guangzhou, China). The Turbofect transfection reagent (Thermo Fisher Scientific, USA) was used for plasmid transfection based on provided directions. For in vivo research, a short hairpin RNA (shRNA) construct was used to stably knock down LINC01016 and was expressed with the pGPU6/GFP/Neo vector (GenePharma, Shanghai, China). Sequences for primers and oligonucleotides were listed in Supplementary Table 1 .
9
3D Culture of MCF10A Breast Cells
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The MCF10A human breast epithelial cells were the gift of Dr. Ben Ho Park, Johns Hopkins University School of Medicine. The MCF10A cell line [14] (link) is commercially available from ATCC (Manassas, VA). MCF10A cells were maintained in DMEM/F12 supplemented with 5% heat-inactivated horse serum, 20 ng/ml epidermal growth factor, 0.5 µg/ml hydrocortisone, 100 ng/ml cholera toxin, and 10 µg/ml insulin. Cells were plated in 3D culture essentially as described in [13] (link). Briefly, MCF10A cells were plated onto growth factor reduced Matrigel™ in 8-well chamber slides (5000 cells per well). The day the cells were plated was designated “day 0.” The cultures were fed on days 4, 8, 12, and 16 after plating. For the mixed cell experiment, used to determine if cells can move into adjacent acini, MCF10A cells engineered to express either H2B-mCherry or H2B-GFP were combined and plated in a 1∶1 ratio. The GFP in fixed cultures was detected with a rabbit anti-GFP antibody purchased from Novus Biologicals (Littleton, CO).
10
TGF-β1 Treatment of MCF10A Cells
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The MCF10A cell line was obtained from ATCC (Manassas, VA, USA) and cultured with MEGM (Lonza/Clonetics Corporation, Guangzhou, China) without GA-1000. Cholera toxin (B8326, ApexBio Technology, TX, USA) was added to a final concentration of 100 ng/mL. MCF10A cells were treated for 3 days with TGF-β1 (5 or 10 ng/mL, PHG9214, Life Technologies, Beijing, China).
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