Human embryonic kidney 293 T cells were used for the production of control or mouse CTRP3-enriched conditioned media. Cells were cultured in DMEM containing 10% FBS and 1% antibiotics-antimycotics. Cells were transfected with pCMV14-empty or pCMV14-Ctrp3-3xflag vectors using Opti-MEM and polyethylenimine transfection reagent (Sigma-Aldrich). After 24 hours, culture medium was changed to serum-free DMEM containing 1% antibiotics-antimycotics. After another 24 hours, culture medium was collected and centrifuged at 425g for 20 min at 4°C. The supernatant medium was harvested and filtered through a 0.22-μm filter to remove cell debris. Abundant secretion of CTRP3 into CTRP3-conditioned media was confirmed by immunoblotting.
Polyethylenimine transfection reagent
Manufactured by Merck Group
Polyethylenimine (PEI) is a cationic polymer used as a transfection reagent for the delivery of nucleic acids into eukaryotic cells. It functions by forming complexes with DNA, RNA, or other negatively charged molecules, which can then be internalized by the cells.
Automatically generated - may contain errors
4 protocols using polyethylenimine transfection reagent
1
Production of CTRP3-enriched Conditioned Media
2
Investigating VEGF-C Promoter Regulation
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CAL33 cells belonging to the CR-MI group were transfected by using 50 μl NaCl buffer, 1.25 μl of polyethylenimine transfection reagent (Sigma Aldrich) and 0.5 μg of total test plasmid DNA-renilla luciferase. The plasmids encoded either (i) a human vegf-c promoter fragment with either a non-mutated (wild type, WT) or a mutated (MUT) binding site for the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB),32 (link) (ii) an artificial promoter containing three binding sites for human NF-κB or (iii) a human VEGF-C 3′UTR reporter (LightSwitch, S803537, Active Motif, Carlsbad, CA, USA), all cloned downstream of the luciferase reporter gene. A CMV plasmid was used to control the variability of transfection efficiency in the reporter assays.
3
Transfection Methods for Cell Lines
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HEK-293A cells were transfected by combining 8 μg of plasmid DNA and 32 μL of polyethylenimine transfection reagent (Sigma). Murine fibroblasts were transfected using Lipofectamine 2000 (ThermoFisher, Dreieich, Germany) according to the manufacturer’s protocol. For virus reconstitution, NIH-3T3 cells were transfected using 3 μg of BAC DNA and 10 μL Polyfect (Qiagen, Hilden, Germany).
4
Wnt Signaling Pathway Luciferase Assay
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Transient transfections were performed with polyethylenimine transfection reagent (Sigma, St. Louis, MO) on 1×105 H460 and A549 cells that were plated in a 24-well plate. In the corresponding wells, 0.5 μg of the TOP-FLASH or FOP-FLASH firefly luciferase reporter plasmid and, as an internal control, 0.05 ug of the Renilla luciferase reporter pTK (Promega, Madison, WI) was used. After 24 h, DMSO as a control or 10nM MRx102 was added to the corresponding wells. After 48 h of treatment, the cell lysate was collected and the luciferase activity was determined using the Dual Luciferase Assay System (Promega, Madison, WI) and a luminonmeter. The firefly luciferase activity was normalized to the Renilla luciferase activity.
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