All experiments were done using MCF-10A cells, a non-transformed human mammary epithelial cell line, unless otherwise noted, and were obtained from ATCC (CRL-10317). In addition to MCF10A cells, APC activity was also measured in both HeLa (ATCC, CCL-2) and BJ-5ta cells (ATCC, CRL-4001).
Mcf10a cells
Manufactured by American Type Culture Collection
Sourced in United States, United Kingdom, Germany
The MCF10A cells are a non-tumorigenic human breast epithelial cell line. They are commonly used as a model system for studying normal breast epithelial cell biology.
Automatically generated - may contain errors
Lab products found in correlation
Cholera toxin, by Merck Group (41 mentions)
Hydrocortisone, by Merck Group (40 mentions)
Horse serum, by Thermo Fisher Scientific (34 mentions)
Insulin, by Merck Group (33 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (32 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (25 mentions)
Dmem f12, by Thermo Fisher Scientific (25 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (19 mentions)
Mda mb 231, by American Type Culture Collection (18 mentions)
Epidermal growth factor (egf), by Thermo Fisher Scientific (17 mentions)
Penicillin, by Thermo Fisher Scientific (16 mentions)
Streptomycin, by Thermo Fisher Scientific (16 mentions)
Epidermal growth factor (egf), by Merck Group (15 mentions)
Mda mb 231 cell, by American Type Culture Collection (14 mentions)
Mcf 7, by American Type Culture Collection (13 mentions)
Mcf 7 cell, by American Type Culture Collection (13 mentions)
Dmem f12, by Merck Group (7 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (6 mentions)
Penicillin, by Merck Group (6 mentions)
Mda mb 468, by American Type Culture Collection (6 mentions)
180 protocols using mcf10a cells
1
Measuring APC Activity in Cell Lines
2
Cell Culture Protocols for Common Cell Lines
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BEAS-2B cells were purchased from ATCC (Teddington, UK) and cultured in keratinocyte complete medium, containing 0.0317 mg/mL EGF Human Recombinant, 15.7 mg/mL Bovine Pituitary Extract. Human breast epithelial (MCF10A) cells were purchased from ATCC (Teddington, UK). A549 cells, MCF10A cells, MCF-7 cells and MDA-MB-231 cells were purchased from ATCC (Teddington, UK) and cultured in complete medium; (DMEM, Sigma-Aldrich, UK), 10% (v/v) FBS (BioSera, UK), 1% (v/v) L-glutamine (Sigma-Aldrich, UK), 1% (v/v) penicillin/streptomycin (Invitrogen, UK). HEK293 cells were purchased from ATCC (Teddington, UK) and cultured in complete medium (EMEM, 1X; Thermo Fisher Scientific, UK), 10% (v/v) FBS, 1% (v/v) L-glutamine. H1975 cells were cultured in Roswell Park Memorial Institute Medium (RPMI-1640; L-glutamine, phenol red, Sigma-Aldrich, UK), 10% (v/v) FBS, 1% (v/v) L-glutamine. Cells were incubated at 37 °C and 5% (v/v) CO2 and were passaged at a confluency of >80% within tissue culture flasks with a surface area of 25 cm2 (10 mL, Thermo Scientific, USA) using Accutase (BioLegend UK Ltd, UK) to detach cells. Cell lines were tested for mycoplasma contamination monthly using PCR methodology.
3
Culturing Breast Epithelial Cell Lines
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Human breast epithelial T47D cells used in this study were cloned from a population originally obtained from Dr. G. Green (U. Chicago). T47D cells were routinely tested to ascertain their estrogen-sensitivity before performing experiments with them47 (link). These cells were grown in DMEM containing 5% FBS (propagation medium). When looking at effects of hormones on these cells using 2D culture experiments, we used DMEM/F12 without phenol red with 5% charcoal-dextran stripped FBS (CD-FBS) and 106 U/ml penicillin. When performing 3D culture experiments with the same purpose, we used a mixture of 75% DMEM, 25% Ham’s F12 without phenol red, 7.5% CD-FBS and 2 mM L-glutamine. Human breast epithelial MCF10A cells were purchased from American Type Culture Collection (Manassas, VA) and maintained in DMEM/F12 with phenol red, 5% equine serum, 20 ng/ml EGF, 0.5 µg/ml hydrocortisone, 0.1 µg/ml cholera toxin, and 10 µg/ml insulin. Experiments performed using MCF10A cells used the same medium. In all cases, cells were incubated at 37 °C in 6% CO2 and 100% humidity.
4
Culturing MCF10A and HEK293T Cell Lines
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MCF10A cells were obtained from the American Type Culture Collection (ATCC, CRL-10317). HEK293T cells were obtained from D Ron. Cells were maintained in a 37°C humidified 5% CO2 ventilator and passaged every 2–4 days depending on the cell line. MCF10A cells were grown in MCF10A culture medium (1:1 DMEM:Ham’s F12, supplemented with 5% [v/v] horse serum, 20 ng/ml EGF, 0.5 mg/ml hydrocortisone, 100 ng/ml cholera toxin, and 10 mg/ml insulin) (Debnath et al., 2003 (link)). PTPRK-KO MCF10A cells and Doxycycline-inducible stable cell lines were generated in a previous study (Fearnley et al., 2019 (link)) and cultured as for WT cells. HEK293T cells were cultured in DMEM containing 10% (v/v) FBS (Sigma-Aldrich), 2 mM l -glutamine (Sigma-Aldrich). For Doxycycline induction of MCF10A cell lines, 2 × 106 were seeded per 10 cm dish and cultured for 6 days, with media changes on days 2 and 4. Doxycycline (Sigma-Aldrich) was included on day 4, 48 hr prior to lysis. Cell lines that were not obtained from commercial sources were subjected to Mycoplasma testing using MycoAlert PLUS (Lonza) or MycoProbe (R&D Systems) Mycoplasma Detection Kits.
5
Cell line culture and maintenance
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Retinal pigmented epithelium (RPE1) cells, human mammary epithelial MCF10A cells, human mammary breast BT-20 cells, and MDA-MB-231 cells were purchased from the American Type Culture Collection (ATCC, Wesel, Germany). RPE1 p53 knockout cells were described earlier (Zomerman et al. 2018 (link)). RPE1, BT-20, and MDA-MB-231 cells were cultured in Dulbecco's Modified Eagle Medium (DMEM; Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA, USA) and 50 U/µl Penicillin/Streptomycin solution. MCF10A cells were cultured in DMEM/F-12 (Gibco, Carlsbad, CA, USA) supplemented with 5% horse serum (Invitrogen, Carlsbad, CA, USA), 50 U/µl Penicillin/Streptomycin, 100 ng/ml cholera toxin (Sigma), 20 ng/ml epidermal growth factor (Peprotech, Rocky Hill, CT, USA), 10 µg/ml insulin (Sigma), and 500 ng/ml hydrocortisone (Sigma).
6
Culturing Diverse Mammalian Cell Lines
7
MCF-10A Cell Culture and Characterization
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MCF-10A cells were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). Phenol red-free DMEM/F-12 media, horse serum, penicillin/streptomycin, antibiotic/antimycotic, human insulin (Novolin R), epidermal growth factor, trypsin-EDTA (10X), Hanks Balanced Salt Solution (HBSS), and Phosphate Buffered Saline (PBS) were purchased from Invitrogen (Carlsbad, CA, USA). Cholera toxin was purchased from Enzo Life Sciences (Plymouth Meeting, PA, USA). The CellTiter 96® AQueous One Solution Cell Proliferation Assay was purchased from Promega (Madison, WI, USA). The Bromodeoxyuridine Cell Proliferation (Chemiluminescent) Assay kit was purchased from Cell Signaling Technology (Danvers, MA, USA). The EpiQuik 8-OHdG DNA Damage Quantification Direct Kit (Colorimetric) was purchased from EpiGentek (Farmingdale, NY, USA). The Qiagen Genomic-tip 20/G, Genomic DNA buffer set, and proteinase k were purchased from Qiagen (Germantown, MD, USA). benzo(a)pyrene (BaP), diallyl sulfide (DAS), PeroxiDetectTM Kit, and all other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies were purchased from Cell Signaling (GAPDH (D16H11) Rabbit mAb; #5174S) and Abcam (DNA Polymerase β; ab26343).
8
Cell Culture and Transfection Protocol
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We originally purchased MCF7 and MCF10A cells from American Type Culture Collection. We maintained the MCF7 and Hela cells in Dulbecco’s modified Eagle’s medium (DMEM) (Corning) media, supplemented with 10% fetal bovine solution (FBS) (GenDEPOT) and maintained the MCF10A cells in DMEM/F-12 (Corning) supplemented with 5% horse serum, 20 ng ml−1 EGF, 0.5 mg ml−1 hydrocortisone, 100 ng ml−1 cholera toxin, 10 μg ml−1 insulin in a 5% CO2 incubator at 37 °C4 (link),65 (link). Transfection of LNA GapmeRs (Qiagen) into the cells was carried out using Lipofectamine 2000 (Life Technologies) according to the manufacturer’s protocol and at a final concentration of 60 nM. For NET1e eRNA knockdown, a mixture of NET1e LNA 1, 2, and 3 was transfected into the cell. The sequence information for LNA is described in Supplementary Table 2 .
9
Cell Culture Protocols for Cancer Research
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The adopted cells (breast cancer MCF-7 and MDA-MB-231 and human mammary epithelial MCF-10A cells) were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured as indicated [18 (link)]. The mouse embryonic fibroblast BALB/3T3 and neuroblastoma SH-SY5Y were also obtained from American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in DMEM high glucose supplemented with 100 U mL−1 penicillin/streptomycin and 10% bovine calf serum (BCS) or 10% fetal bovine serum (FBS), respectively.
10
Establishment and Characterization of MCF-10A v-Src:ER Cell Line
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MCF-10A cells are immortalized normal human mammary epithelial cells that do not express the estrogen receptor (ER) [27 (link),29 (link)]. MCF-10A cells were obtained from the American Type Culture Collection (ATCC). We generated MCF-10A cells stably expressing v-Src:ER chimeras (MCF-10A v-Src:ER) by using retroviruses and transformed them with 1 μM 4-Hydroxytamoxifen (H7904; Sigma) or the solvent ethanol as a control. MCF-10A cells were cultured in Assay Medium. Assay Medium is DMEM/F12 media (1:1) (11320–033; Gibco) supplemented with 2% horse serum (16050–122; Gibco), 0.5 μg/ml hydrocortisone (H4001; Sigma), 10 μg/ml insulin (093–06351; Wako), 100 ng/ml Cholera toxin (C8052; Sigma) and penicillin-streptomycin (15140–122; Gibco). In both siRNA knockdown experiments and Soft Agar Colony Formation Assays, Growth Medium was used as culture medium. Growth Medium is DMEM/F12 media (1:1) supplemented with 5% horse serum, 0.5 μg/ml hydrocortisone, 10 μg/ml insulin, 20 ng/ml epidermal growth factor (AF-100–15; PeproTech), 100 ng/ml Cholera toxin and penicillin-streptomycin. MDA-MB-231 cells, which are malignant mammary cancer cells, were cultured in DMEM/F12 media (1:1) supplemented with 10% FBS and penicillin-streptomycin. All cell lines were incubated at 37°C in a humidified atmosphere containing 5% CO2.
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