The MCF7 Tet-On Advanced cell line was obtained from Clontech. The generation and characterization of the MCF7GFP-IBD cell line has been described (24 (link)) and was used with further authentication by IDEXX BioResearch within the last 6 months. Panc 02GFP-IBD, U-87 MGGFP-IBD, and hTERT-HME1GFP-IBD cell lines were developed similarly from parent cell lines purchased from American Type Culture Collection (ATCC). Briefly, GFP-IBD cloned into the pLVX-Tight-Puro vector was transfected along with pLVX-Tet-On Advanced vector (Clontech) into each cell line. Following G418 and puromycin selection, cells were induced with 1 μg/mL doxycycline and sorted to establish the IRIF reporter cell lines. The Panc 02GFP-IBD and U-87 MGGFP-IBD cell lines were maintained in RPMI (Invitrogen), supplemented with 10% Tet system approved FBS (Clontech). The hTERT-HME1GFP-IBD cell line was maintained in MEBM media supplemented with MEGM SingleQuot (Lonza). For studies requiring glucose and glutamine limitation, media was prepared using DMEM base, D-glucose (Sigma), and L-glutamine solutions (Gemini Bioproducts) at appropriate concentrations with 10% FBS.
Megm singlequot
Manufactured by Lonza
Sourced in United States
MEGM SingleQuots is a kit that provides aliquoted supplements for the Mammary Epithelial Growth Medium (MEGM) formulation. The components in the kit are pre-measured and ready-to-use, facilitating the preparation of the complete growth medium.
Automatically generated - may contain errors
Lab products found in correlation
Mda mb 231, by American Type Culture Collection (6 mentions)
Cholera toxin, by Merck Group (5 mentions)
Mcf 10a, by American Type Culture Collection (5 mentions)
Cholera toxin, by Lonza (5 mentions)
Mcf 10a, by Lonza (4 mentions)
Mcf 7, by American Type Culture Collection (4 mentions)
Mammary epithelium basal medium, by Lonza (3 mentions)
Medium 199, by Merck Group (3 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)
Hcc1937, by American Type Culture Collection (2 mentions)
Bt549, by American Type Culture Collection (2 mentions)
Ga 1000, by Lonza (2 mentions)
Hygromycin b, by Thermo Fisher Scientific (2 mentions)
Sodium pyruvate, by Corning (2 mentions)
Dulbecco modified eagle medium (dmem), by Lonza (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
L glutamine, by Thermo Fisher Scientific (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Tet system approved fbs, by Takara Bio (1 mentions)
Plvx tight puro vector, by Takara Bio (1 mentions)
28 protocols using megm singlequot
1
Development of GFP-IBD Reporter Cell Lines
2
Culturing Breast Cancer and Control Cell Lines
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The MDA-MB-231, HCC1937, T47D, BT549, MCF-7, and HEK293 cell lines and the human mammary epithelial cell line MCF-10A were purchased from the American Type Culture Collection (ATCC). Cells were grown in DMEM (MDA-MB-231, MCF-7, HCC1937, and HEK293) or RPMI (T47D and BT549) supplemented with 10% FBS. MCF-10A cells were maintained in MEBM (Lonza) supplemented with 100 ng/mL cholera toxin (Sigma) and MEGM SingleQuot (Lonza) supplemented with GA-1000 (a gentamycin–amphotericin B mixture). All cell lines were maintained at 37°C in a humidified atmosphere of 5% CO2 before experiments. The Lipofectamine 2000 transfection reagent (Invitrogen) was used to obtain stable GPR1-overexpressing HEK293 cells (HEK293 GPR1+/+ cells), and the pcDNA3.0 empty vector was used as a control (HEK293 pcDNA cells).
3
Cell Culture Maintenance of TNBC Lines
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The TNBC cell lines MDA-MB-231 and HCC1937, and the human mammary epithelial cell line MCF-10A were purchased from the American Type Culture Collection (ATCC). Cells were grown in DMEM (MDA-MB-231, HCC1937) supplemented with 10% FBS. MCF-10A cells were maintained in MEBM (Lonza) supplemented with 100 ng/mL cholera toxin (Sigma) and MEGM Single Quot (Lonza) supplemented with GA-1000 (gentamycin-amphotericin B mixture). All cell lines were maintained at 37 °C in a humidified atmosphere of 5% CO2 before experiments.
4
Cell Culture and Proliferation Assay
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MCF10A cells were cultured in MEBM (Lonza) containing 52 μg/ml Bovine Pituitary Extract (BPE), 500 ng/ml hydrocortisone, 10 ng/ml hEGF, 5 μg/ml insulin (MEGM SingleQuot, Lonza) and 100ng/ml cholera toxin (Wako). MCF-7, MDA-MB-231, A549, and HT1080 cells were maintained in Dulbecco's modified Eagle (DME) medium supplemented with 10% fetal bovine serum (GM). We obtained TGFβ1 from R&D systems iCRT3 from Sigma-Aldrich, FH535, PD98059 and LY294002 from Calbiochem, and flavone (2-phenylchromone) from Nacalai Tesque. Cell proliferation was estimated using the colorimetric cell counting reagent WST-8 (SF reagent; Nacalai Tesque) in triplicate cultures.
5
Oncosphere Formation Assay for Tumor Initiation
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To assess tumor initiation capacity in vitro, cells were counted and plated into low-attachment 96-well plates at dilution of 1 cell per well. They were then cultured in mammary epithelial basal medium (MEBM, Lonza, cat no. CC-3151), supplemented with MEGM SingleQuots (which contain Insulin, EGF, Hydrocortisone and GA-1000, LONZA cat no. 4136), 1X B27 without retinoic acid (GIBCO, cat no. 12787-010), and 20 ng/ml of recombinant fibroblast growth factor (GIBCO, cat no. PHG0026), and incubated in 5% CO2, 37°C in order to obtain a first generation of oncospheres (anoikis and pluripotency selection) after 15 days. The process was repeated to ensure second-generation oncospheres (pluripotency selection). After 2 weeks of culture, the oncospheres were counted under the microscope.
6
Culturing Breast Cancer Cell Lines
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The cell lines, MCF-7, MDA-MB-231, and MCF10A, were obtained from American Type Culture Collection (ATCC). MCF-7 and MDA-MB-231 cells were maintained in DMEM growth medium with 10% fetal bovine serum (FBS, Hyclone) and 100 IU/mL penicillin-streptomycin (Invitrogen). For MCF-7 only, 0.01 mg/mL human recombinant insulin (Sigma) was also added. MCF10A were maintained in MEBM basal medium supplemented with MEGM SingleQuots (Lonza). All cell lines were maintained at 37°C under 5% humidified CO2.
7
Cell Culture Protocols for HEK293T, NIH3T3, BJ-5ta, and MCF10A
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Cell Culture HEK293T Phoenix (National Gene Vector Biorepository, Indianapolis, IN, USA) and NIH3T3 (ATCC; CRL-1658) cell lines were cultured in DMEM with 4.5g/L glucose, L-glutamine, and sodium pyruvate (CORNING) and supplemented with 10% (v/v) fetal bovine serum (FBS; Hyclone). BJ-5ta cell lines were cultured in DMEM supplemented with a 4:1 ratio of Medium 199 (Sigma-Aldrich) and 0.01 mg/ml hygromycin B (Invitrogen) and 10% (v/v) FBS. MCF10A (ATCC; CRL-10317) cell lines were cultured in Mammary Epithelium Basal Medium (Lonza) supplemented with MEGM SingleQuots (Lonza) with the following modification: gentamicin sulfateamphotericin was omitted from media, and cholera toxin (Sigma-Aldrich) was added at a final concentration of 100 ng/ml. All cells were grown at 37°C with 5% CO2 in a humidified incubator.
8
Breast Cancer Cell Culture Protocol
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MDA-MB-231 (ATCC Cat. HTB-26), 4T1, 4TO7, 66c14 cells were cultured in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin. The 4TO7, 66c14, and 4T1 cell lines were kindly provided by Dr. Yibin Kang at Princeton University. HMEC cells (ATCC Cat. PCS-600-010) were cultured in MEGM (Lonza CC-3151) supplemented with MEGM SingleQuots (Lonza CC-4135). MCF7-Ras cells were cultured in DMEM/F-12 supplemented with10% FBS and 1% penicillin/streptomycin. MCF10 and derived cell lines were kindly provided by Dr. Lalage M. Wakefield at the National Cancer Institute and cultured as described in [30 (link)]. All cells were cultured at 37°C, 5% CO2.
9
Cell Culture Conditions for Diverse Cell Lines
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NIH3T3 and HEK293T Phoenix cell lines were cultured in DMEM with 4.5 g/liter glucose, l -glutamine, and sodium pyruvate (Corning). BJ-5ta cell lines were cultured in DMEM supplemented with a 4:1 ratio of Medium 199 (Sigma-Aldrich) and 0.01 mg/ml hygromycin B. MCF10A cell lines were cultured in Mammary Epithelium Basal Medium (Lonza) supplemented with MEGM SingleQuots (Lonza) with the following modification: gentamicin sulfate-amphotericin was omitted from media, and cholera toxin (Sigma-Aldrich) was added at a final concentration of 100 ng/ml. All media were supplemented with 10% (vol/vol) FBS (Hyclone) at 37°C with 5% CO2 in a humidified incubator.
10
Culturing Breast Cancer Cell Lines
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All cell samples were cultured as described previously by Meksiarun et al.12 (link)
Four types of human epithelial breast tumour cells (MCF-7, BT-474, MDA-MB-231, and SK-BR-3) were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS, Hyclone). MCF10A, the normal breast cells were cultured in MEBM medium supplemented with MEGM SingleQuots (Lonza, Basel, Switzerland). Five percent CO2 was supplied to the cells in an incubator, and the temperature was maintained at 37.8 ℃. The cells were harvested and plated into 12 identical flasks as equivalent aliquots of cell suspension. The flask cultures were incubated for 96 h to reach approximately 50% confluency. Fresh media were replaced in the cultures 1 h before irradiation. All flasks were removed from the incubator for less than 40 min before irradiation.
Four types of human epithelial breast tumour cells (MCF-7, BT-474, MDA-MB-231, and SK-BR-3) were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS, Hyclone). MCF10A, the normal breast cells were cultured in MEBM medium supplemented with MEGM SingleQuots (Lonza, Basel, Switzerland). Five percent CO2 was supplied to the cells in an incubator, and the temperature was maintained at 37.8 ℃. The cells were harvested and plated into 12 identical flasks as equivalent aliquots of cell suspension. The flask cultures were incubated for 96 h to reach approximately 50% confluency. Fresh media were replaced in the cultures 1 h before irradiation. All flasks were removed from the incubator for less than 40 min before irradiation.
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