We obtained the human BC cell lines MDA-MB-231, BT549, MCF-7, SKBR3 and normal breast epithelial cell line MCF-10A from Chinese Academy of Sciences (Shanghai, China). All BC cell lines were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco, USA) with 10% Fetal Bovine Serum (FBS) (Gibco, USA), penicillin (100 units/ml) and streptomycin (100 μg/ml) (Enpromise, China), and mycoplasma elimination reagent (Yeasen, China). MCF-10A were cultured in Mammary Epithelial Basal Medium (MEBM) (Cambrex, USA). All these cells were cultured at 37°C with 5% CO2. MiR-640 mimics, miR-640 inhibitor and non-specific miR-negative control (miR-640-NC) oligo were purchased from RiboBio (Guangzhou, China). The specific siRNA of Wnt7b (si-Wnt7b) and negative control (si-NC) were purchased from Generay (Shanghai, China). Hieff Trans™ Liposomal Transfection Reagent (Yeasen, China) was used for transfection according to the protocols.
Mammary epithelial basal medium
Manufactured by Cambrex
Sourced in United States
Mammary Epithelial Basal Medium (MEBM) is a cell culture medium designed to support the growth and maintenance of mammary epithelial cells. It provides a balanced formulation of nutrients, growth factors, and other components necessary for the in vitro culture of these cell types.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (5 mentions)
Mda mb 231, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Dna midiprep kit, by Qiagen (2 mentions)
Mcf 7, by Thermo Fisher Scientific (2 mentions)
Hcc1937, by Thermo Fisher Scientific (2 mentions)
Mcf 10a, by Cambrex (2 mentions)
Hieff trans liposomal transfection reagent, by Yeasen (1 mentions)
Mycoplasma elimination reagent, by Yeasen (1 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions)
Mir 200a 3p mimic, by RiboBio (1 mentions)
Mir 200a 3p inhibitor, by RiboBio (1 mentions)
Penicillin streptomycin p s, by Merck Group (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Gibco dulbecco s modified eagle medium dmem, by Thermo Fisher Scientific (1 mentions)
6 protocols using mammary epithelial basal medium
1
Breast Cancer Cell Line Characterization
2
Investigating Breast Cancer Cell Lines
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The human BC cell lines MDA-MB-231, MCF-7, HCC-1937, SKBR3 and normal breast epithelial cell line MCF-10A were purchased from Chinese Academy of Sciences (Shanghai, China). MDA-MB-231, MCF-7, HCC-1937 and SKBR3 cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco, USA) with 10% Fetal Bovine Serum (FBS) (Gibco, USA), penicillin (100 units/ml) and streptomycin (100 μg/ml) (Enpromise, China). MCF-10A cells were cultured in Mammary Epithelial Basal Medium (MEBM) (Cambrex, USA). All these cells were cultured at 37 °C with 5% CO2. Small, interfering, specifically targeting human hsa_circ_0005273 (si-circ_0005273), non-specific negative control oligos (si-NC) and hsa_circ_0005273 lentiviral plasmid (lv-circ_0005273) were purchased from IBSBio (Shanghai, China). Human miR-200a-3p-mimics, non-specific negative control (miR-200a-3p-NC) and miR-200a-3p inhibitor were purchased from RiboBio (Guangzhou, China). MDA-MB-231, MCF-7 and SKBR3 cells were cultured and transfected with reagents above using Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, USA) according to the manufacturer’s instructions. We used DNA Midiprep Kits (Qiagen, Hilden, Germany) to prepare plasmid vectors. A lentivirus carrying si-circ_0005273 was constructed by ZORIN (Shanghai, China) and transfection procedures were performed according to the manufacturer’s instructions.
3
Breast Cancer Cell Line Culture
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The human HEK293T and human BC cell lines (MDA-MB-231, MCF-7, HCC-1937, and SKBR3) and normal breast epithelial cell line (MCF-10A) were obtained from Chinese Academy of Sciences (Shanghai, China). The HEK293T, MDA-MB-231, MCF-7, HCC-1937, and SKBR3 cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) (Gibco, USA) with 10% Fetal Bovine Serum (FBS) (Gibco, USA), penicillin (100 units/ml), and streptomycin (100 μg/ml) (Enpromise, China). The MCF-10A cells were cultured in Mammary Epithelial Basal Medium (MEBM) (Cambrex, USA). All cells were cultured at 37°C with 5% CO2.
4
Breast Cancer Cell Line Culture and Manipulation
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The human BC cell lines MDA-MB-231, MCF-7, HCC-1937, SKBR3 and normal breast epithelial cell line MCF-10A were purchased from Chinese Academy of Sciences (Shanghai). MDA-MB-231, MCF-7, HCC-1937 and SKBR3 cells were cultured in Dulbecco's Modi ed Eagle's Medium (DMEM) (Gibco, USA) with 10% Fetal Bovine Serum (FBS) (Gibco), penicillin (100 units/ml) and streptomycin (100 µg/ml) (Enpromise, China). MCF-10A cells were cultured in Mammary Epithelial Basal Medium (MEBM) (Cambrex). All these cells were cultured at 37 ℃ with 5% CO2. Small, interfering, speci cally targeting human hsa_circ_0005273 (si-circ_0005273), non-speci c negative control oligos (si-NC) and has_circ_0005273 overexpression(circ_0005273), were purchased from IBSBio(Shanghai, China).
Human miR-200a-3p-mimics and the corresponding negative control mimics (miR-200a-3p-NC) and miR-200a-3p inhibitor were purchased from RiboBio(Guangzhou, China). MDA-MB-231 and MCF-7 cells were cultured and transfected with reagents above using Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scienti c, Inc.) according to the manufacturer's instructions for 6 h at 37 °C for transient transfection. We used DNA Midiprep Kits (Qiagen, Hilden, Germany) to prepare plasmid vectors. A lentivirus carrying si-circ_0005273 was constructed by ZORIN(Shanghai, China) and transfection procedures were performed according to the manufacturer's instructions.
Human miR-200a-3p-mimics and the corresponding negative control mimics (miR-200a-3p-NC) and miR-200a-3p inhibitor were purchased from RiboBio(Guangzhou, China). MDA-MB-231 and MCF-7 cells were cultured and transfected with reagents above using Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scienti c, Inc.) according to the manufacturer's instructions for 6 h at 37 °C for transient transfection. We used DNA Midiprep Kits (Qiagen, Hilden, Germany) to prepare plasmid vectors. A lentivirus carrying si-circ_0005273 was constructed by ZORIN(Shanghai, China) and transfection procedures were performed according to the manufacturer's instructions.
5
Culturing and Transfecting Breast Cell Lines
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All BC cell lines (MDA-MB-231 and BT-549) and normal breast epithelial cell lines (MCF-10A) were acquired from Chinese Academy of Sciences (Shanghai, China). All cell lines were authenticated and tested for mycoplasma contamination. The BC cell lines were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco, USA) with 10% Fetal Bovine Serum (FBS, Gibco, USA) and 1% penicillin-streptomycin (PS, Sigma, Germany). MCF-10A were cultured in Mammary Epithelial Basal Medium (MEBM) (Cambrex, USA). All cells were cultured in a 5% CO2 incubator at 37 °C. Hieff TransTM Liposomal Transfection Reagent (Yeasen, China) was used for transfection following the manufacturer’s instructions. Small interfering RNA targeting on circPRKCI (si-circPRKCI) and the negative control (si-NC) were purchased from IBSbio (Shanghai, China). Inhibitors, mimics and the negative control (miR-NC) for miR-545-3p were purchased from RiboBio (Guangzhou, China). Lentiviral plasmid for overexpressing circPRKCI (LV-circPRKCI) and the negative control (LV-vector) were designed by QiheBio (Shanghai, China).
6
Breast Cancer Tissue Collection and Cell Culture
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Tissue samples were obtained from patients with BC who underwent radical mastectomy at Shanghai Tenth People’s Hospital (Shanghai, China). A total of 30 pairs of tumor and matched normal adjacent breast tissues were collected and preserved in liquid nitrogen. All clinical samples were obtained with the written informed consent of participants, and the project was approved by the Ethics Committee of Shanghai Tenth People’s Hospital (No. 2020-KN174-01). BC cells (MDA-MB-231, BT549, and MCF-7) and mammary epithelial cells (MCF-10A) were purchased from the Cell Bank of Type Culture Collection of Chinese Academy and grown in mammary epithelial basal medium (Cambrex, East Rutherford, NJ, USA) and Gibco Dulbecco’s modified Eagle medium (DMEM; Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco) and 1% penicillin-streptomycin (Enpromise, Shanghai, China). All cells were maintained in a 5% CO2 incubator at 37 °C.
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