The MDA-MB-231 and MDA-MB-468 cells at a density of 3 × 105 were transfected using Lipofectamine® 2000 transfection reagent (Invitrogen; Thermo Fisher Scientific, Inc.). The negative control (NC) and FEN1 siRNA sequences from Guangzhou RiboBio Co., Ltd., (Guangzhou, China) were as follows: NC forward, 5′-UUCUCCGAACGUGUCACGUTT-3′ and reverse, 5′-ACGUGACACGUUCGGAGAATT-3′; FEN1 forward, 5′-GGGUCAAGAGGCUGAGUAAdTdT-3′ and reverse, 5′-dTdTCCCAGUUCUCCGACUCAUU-3′. The NC or FEN1 siRNA (10 nM) and Lipofectamine® 2000 were diluted in L-15 medium. Following 20 min of incubation at 37°C, the complexes were added to each well of 6-well plates containing serum-free L-15 and cells. Following 72 h of transfection, cells were used in the subsequent experiments.
Lipofectamine
Manufactured by RiboBio
Sourced in China
Lipofectamine® is a cationic lipid-based transfection reagent used for the delivery of nucleic acids, such as plasmid DNA, siRNA, and mRNA, into eukaryotic cells. It facilitates the uptake and expression of these molecules within the target cells. The product is designed to improve the efficiency of transfection while minimizing cytotoxicity.
Automatically generated - may contain errors
5 protocols using lipofectamine
1
Transfection of Breast Cancer Cells
2
Investigating PTX3 and JUN in Glioma
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Human GBM cells (U87-MG) are purchased from the Chinese Academy of Sciences. Glioma cells are maintained in DMEM medium with 10% fetal bovine serum and 1% penicillin–streptomycin, 5% CO2 and 37°C. Cells are randomly divided into different groups, the control group, the siRNA-NC (si-NC) group, the siRNA-PTX3 (si-PTX3) group, the overexpression JUN group and the overexpression JUN with siRNA-PTX3 group.
The siRNA of PTX3 (5′-GGTCAAAGCCACAGATGTA-3′) and JUN overexpression plasmid are obtained from RiboBio (Guangzhou, China). Five microliters of siRNA-PTX3 (or 2.5 μg overexpression JUN plasmid) and 5 μl lipofectamine (RiboBio, China) are added into 100 μl serum-free DMEM. Then, 1 ml DMEM is added and the mixed solution is incubated at 37°C for 6 h. The medium is discarded after 72 h and cells are washed by PBS twice for further experiment.
The siRNA of PTX3 (5′-GGTCAAAGCCACAGATGTA-3′) and JUN overexpression plasmid are obtained from RiboBio (Guangzhou, China). Five microliters of siRNA-PTX3 (or 2.5 μg overexpression JUN plasmid) and 5 μl lipofectamine (RiboBio, China) are added into 100 μl serum-free DMEM. Then, 1 ml DMEM is added and the mixed solution is incubated at 37°C for 6 h. The medium is discarded after 72 h and cells are washed by PBS twice for further experiment.
3
Dual-Luciferase Assay for SOX12 miR-342-3p Binding
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A Lipofectamine™ 2000 kit was utilized to cotransfect wild-type (WT) or mutant (MUT) DLR vectors (WT-SOX12 or MUT-SOX12; RiboBio, Guangzhou, China) and miR mimics or mimics NC into KG-1 cells and to cotransfect WT-SOX12 or MUT-SOX12 and miR inhibitors or inhibitors NC into HL-60. Luciferase activity was determined 48 h later under the instructions of the DLR gene detection kit (Promega, Madison, WI, USA). The ratio of the luminescence intensity of ranilla luciferase/firefly luciferase was the binding intensity of SOX12 to miR-342-3p.
4
Transfection of miRNA Mimics and Plasmids
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For transfection, cells were first washed once with serum-free medium and then incubated in 4 mL of serum-free medium for 4-6 h. miRNA mimic (100 nM) or other constructs (1 µg/mL for PFL and shRNA-PFL) and lipofectamine 2000 (Invitrogen, Carlsbad, CA) were separately mixed with 500 μL of Opti-MEM ® I Reduced Serum Medium (Gibco, Grand Island, NY) for 5 min. Then, the two mixtures were combined and incubated at room temperature for 20 min. The lipofectamine:miRNA (or plasmid) mixture was added to the cells and incubated in 6-well culture plates at 37°C for 6 h. Let-7d mimic was purchased from RiboBio Co., Ltd. (Guangzhou, China). Subsequently, 5 mL of fresh medium containing 10% FBS was added to the flasks and the cells were maintained in the culture medium for 48 h until the subsequent experiments.
5
Investigating miR-449a-5p Regulation in Cardiomyocytes
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The plasmid pcDNA-CDK6 and pcDNA-NC was synthesized by Vigene. miR-449a-5p mimics, mimic-NCs (cat. no. miR1N0000001-1-5), miR-449a-5p inhibitors, and inhibitor-NCs (cat. no. miR2N0000001-1-5) were synthesized by Guangzhou Ribobio Co., Ltd. The sequences of miR-449a-5p mimics were 5′-UGGCAGUGUAUUGUUAGCUGGU-3′. The sequences of miR-449a-5p inhibitors were 5′-ACCAGCUAACAAUACACUGCCA-3′. After isolated P1 CMs were cultured for 48 h at 37°C, 5 µl Lipofectamine® 2000 and 50 nM plasmids, mimics or inhibitors were added to Opti-MEM. The mixture was added to the cells following incubation at room temperature for 20 min. This medium was replaced after 6 h at 37°C with the same volume of DMEM/F12 medium. RNA or protein was isolated and immunofluorescence analysis was conducted following 48 h.
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