MCF‐7 and MDA‐MB‐231 breast cancer cells were purchased from ATCC/LGC Promochem. Cells were stably transfected as described25 with a pcDNA3.1 control plasmid (Invitrogen) or a plasmid allowing for expression of the open reading frame (1104 bp) of human HS2ST1 (NCBI Reference Sequence: NM_012262) in the vector pReceiver‐M02 under the control of the cytomegalovirus (CMV) promoter (RZPD/ImaGenes). MDA‐MB‐231 cells were maintained in Dulbecco's Modified Eagle Medium (DMEM) containing 10% FCS, 1% glutamine, 1% penicillin/streptomycin and 800 µg/mL G418 in a humidified atmosphere of 7.5% CO2 in air at 37°C. MCF‐7 cells were cultured in RPMI‐1640 medium containing 10% FCS, 1% glutamine, 1% penicillin/streptomycin and 800 µg/mL G418 in a humidified atmosphere of 5% CO2 in air at 37°C. In some experiments, 10 µmol/L U0126 (Cell Signaling Technologies) was used to inhibit the MAPK pathway.
Pcdna3.1 control plasmid
Manufactured by Thermo Fisher Scientific
Sourced in Germany
The PcDNA3.1 control plasmid is a circular DNA molecule that can be used as a control in various molecular biology experiments. The plasmid contains a bacterial origin of replication and an antibiotic resistance gene, allowing for selection and amplification in bacterial cells. The plasmid also includes a eukaryotic promoter and multiple cloning sites, which can be used for the insertion and expression of foreign DNA sequences in mammalian cell lines.
Automatically generated - may contain errors
Lab products found in correlation
Mda mb 231, by LGC (2 mentions)
U0126, by Cell Signaling Technology (1 mentions)
Caco 2, by Thermo Fisher Scientific (1 mentions)
Mcf 7, by LGC (1 mentions)
Fetal calf serum (fcs), by Harvard Bioscience (1 mentions)
Penicillin streptomycin, by Merck Group (1 mentions)
Hela cell line, by LGC (1 mentions)
4 protocols using pcdna3.1 control plasmid
1
Overexpression of HS2ST1 in Breast Cancer Cells
2
Stable Transfection of Caco2 Cells
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The human colon carcinoma cell line Caco2 (German Collection of Microorganisms and Cell Cultures, Department of Human and Animal Cell Cultures, Braunschweig, Germany) was stably transfected with a pcDNA3.1 control plasmid (Invitrogen), or plasmids overexpressing Syndecan-1 cDNA (38 (link)), native HPSE, or enzymatically inactive HPSE double mutated in Glu225 and Glu343 (39 (link)). Stable clones were selected using 800 μg/ml G418. Caco2 cells were maintained in RPMI media containing 10% fetal calf serum (FCS), 1% glutamine, 1% penicillin/streptomycin and 800 μg/ml G418 in a humidified atmosphere of 5% CO2 at 37°C. Successful transfection was confirmed by qPCR.
3
Breast Cancer Cell Lines and Syndecan-1 Overexpression
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MCF-7 and MDA-MB-231 human breast cancer cell lines were purchased from ATCC/LGC Promochem (Wesel, Germany). Cells were cultured as described previously [16 (link)]. Regarding Sdc-1 overexpression, cells were stably transfected with a pcDNA3.1 control plasmid (Invitrogen, Karlsruhe, Germany) or a plasmid enabling the overexpression of wild-type Sdc-1 (Sdc-1-OV) and a constitutively shed variant (392-Sdc-1-OV) of murine Sdc-1 in the vector pReceiver-M02. Using 800 µg/mL of G418, stable clones were selected and characterized [5 (link)].
4
Overexpression of Syndecan-1 in HeLa Cells
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The HeLa cell line was purchased from ATCC/LGC Promochem (Wesel, Germany) and cultured in RPMI (Sigma, cat. No. D8758, Deisenhofen, Germany); containing 10% Fetal Calf Serum (FCS, Biochrom GmbH, Cat. No. S0615, Berlin, Germany) and 1% penicillin/streptomycin (Sigma, cat. No. P433) and maintained in a humidified atmosphere of 5% CO2 at 37°C. Cells were stably transfected with a pcDNA3.1 control plasmid (Invitrogen, Karlsruhe, Germany) or a plasmid allowing for the overexpression of wild-type (WT), a constitutively membrane-bound (Sdc-1-388), and a constitutively shed form (Sdc-1-392) of murine Sdc-1 in the vector pReceiver-M02 under control of the cytomegalovirus promoter (RZPD/ImaGenes, Berlin, Germany) as previously described (10 (link), 20 (link)). Stable clones were selected using 1 mg/ml G418. HeLa cells were cultured in RPMI-1640 medium containing 10% FCS, 1% penicillin/streptomycin and 600 mg/ml G418 in a humidified atmosphere of 5% CO2 at 37°C. Successful transfections were confirmed by qPCR.
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