Dna lipofectamine 2000

Manufactured by Thermo Fisher Scientific

Sourced in United States

DNA-Lipofectamine 2000 is a transfection reagent used for the introduction of DNA into eukaryotic cells. It is a cationic lipid-based formulation that forms complexes with DNA, enabling its delivery into the cells.

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Lipofectamine® 2000 DNA Transfection Reagent Protocol Lipofectamine 2000 DNA Transfection Reagent Lipofectamine 2000 Lipofectamine

6 protocols using dna lipofectamine 2000

SARS-CoV-2 Pseudovirus Production and Characterization

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SARS-CoV-2 pseudovirus (PsV) was produced as described
by Schmidt et al.^{56 (link)} Plasmids containing
the SARS-CoV-2 spike gene [pSARS-CoV1-Strunc, pSARS-CoV2-Strunc (K417N/
E484K/N501Y mutations)], pCRV1NHG GagPol, and pNanoLuc2AEGFP were
used to produce the pseudoviral particle and were kindly provided
by Drs. Theodora Hatziioannou and Paul Bieniasz, Rockefeller University.
The plasmids were used to transfect 293T cells (ATCC, Manassas, VA)
monolayers prepared in six-well plates. Briefly, the DNA/lipofectamine
2000 (Thermo Fisher Scientific, Waltham, MA) mixtures were added to
293T cell monolayers and incubated for 6 h at 37 °C, 5% CO₂, and 98% humidity. The cell monolayers were then washed twice
with D-PBS (Thermo Fisher Scientific) and finally incubated for 48
h at 37 °C, 5% CO₂, 98% humidity in Dulbecco’s
modified Eagle’s medium (DMEM) (Thermo Fisher Scientific) with
10% FBS (Thermo Fisher Scientific) and Penicillin + Streptomycin (Thermo
Fisher Scientific, Waltham, MA). After the 48 h incubation, the cell
supernatants were collected, filtered (using a 0.22 μm pore
size PVDF filter), aliquoted, and stored at −80 °C. The
pseudoviral titer was determined using a cell-based pseudoviral entry
assay^{57 (link)} and the TurboLuc Luciferase One-Step
Glow Assay Kit (Thermo Fisher Scientific).

Rodríguez Y., Cardoze S.M., Obineche O.W., Melo C., Persaud A, & Fernández Romero J.A. (2022). Small Molecules Targeting SARS-CoV-2 Spike Glycoprotein Receptor-Binding Domain. ACS Omega, 7(33), 28779-28789.

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Cell Culture and Transfection Protocols

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Human embryonic kidney HEK293, human cervical cancer HeLa (M-HeLa clone 11), and human adenocarcinomic alveolar basal epithelial A549 cell lines were obtained from the Russian Cell Culture Collection (St. Petersburg, Russia). Non-small-cell lung cancer cell line Calu-1 was kindly provided by Dr. Evgeniy Kopantzev (Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences). The cells were cultured in DMEM/F-12 medium supplemented with 10% fetal bovine serum and 0.3 mg/ml glutamine at 37 °C in humidified atmosphere with 5% CO₂. For transfection, the cells were cultured in 96- or 6-well plates (Corning, USA) for 20–24 h until 70–90% confluence. For transfection plasmid DNA-Lipofectamine 2000 (ThermoFisher Scientific, USA) complexes were prepared following the manufacturer’s protocol in serum-free OptiMEM medium (ThermoFisher Scientific, USA) and then added into the wells; 4 h after the addition of the complexes the medium was replaced with the fresh one.

Komissarov A., Karaseva M., Roschina M., Kostrov S, & Demidyuk I. (2022). The SARS-CoV-2 main protease doesn’t induce cell death in human cells in vitro. PLoS ONE, 17(5), e0266015.

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Multicellular Cytotoxicity Assay Protocol

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Human embryonic kidney HEK293, human cervical cancer HeLa (M-HeLa clone 11), and human adenocarcinomic alveolar basal epithelial A549 cell lines were obtained from the Russian Cell Culture Collection (St. Petersburg, Russia). The cells were cultured in DMEM/F-12 supplemented with 10% fetal bovine serum (ThermoFisher Scientific, Waltham, MA, USA) and 0.3 mg/mL glutamine (Paneco, Moscow, Russia) at 37 °C in a humidified atmosphere with 5% CO₂ in 96- or 6-well plates (Corning, Corning, NY, USA) for 20–24 h until 60–80% confluence. For transfection, plasmid DNA-TurboFect and plasmid DNA-Lipofectamine 2000 (ThermoFisher Scientific, Waltham, MA, USA) complexes were prepared following the manufacturer’s protocol in serum-free OptiMEM (ThermoFisher Scientific, Waltham, MA, USA) and added to the wells; 4 h later, the medium was replaced with the fresh one or with the medium containing necrostatin-1 (5000, 500, 50, 5, or 0.5 μM), PJ34 (50, 5, 0.5, 0.05, or 0.005 μM), cyclosporin A (1000, 100, 10, 1, or 0.1 μM), ferrostatin-1 (200, 20, 2, 0.2, or 0.02 μM), or desferrioxamine (10,000, 1000, 100, 10, or 1 μM). For apoptosis and ferroptosis induction, non-transfected cells were incubated in the growth medium supplemented with 1 μM staurosporine (Sigma-Aldrich, Saint-Louis, MO, USA) or 50 μM erastin (Sigma-Aldrich, Saint-Louis, MO, USA), correspondingly, for 24 h prior to analysis.

Komissarov A.A., Karaseva M.A., Roschina M.P., Shubin A.V., Lunina N.A., Kostrov S.V, & Demidyuk I.V. (2021). Individual Expression of Hepatitis A Virus 3C Protease Induces Ferroptosis in Human Cells In Vitro. International Journal of Molecular Sciences, 22(15), 7906.

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Dual Luciferase Assay for Wnt Signaling

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The dual luciferase assay was performed with the dual-luciferase®reporter assay system (Promega Corp., Madison, WI). All reagents were prepared as described by the manufacturer of the TCF Reporter Plasmid Kit (Millipore Corp., MA, USA). After the transfection complex was formed with FOP DNA, pRL-TK Vector renilla (Promega Corp., Madison, WI) and 50 μl of DNA Lipofectamine™ 2000 (Invitrogen, Carlsbad, CA), the cells were seeded into 96-well plates with 100 μl/well. U2OS cells were divided into four groups: oleandrin (time): 50 nM oleandrin for 0, 24 and 48 h; oleandrin (concentration): 0, 25 and 50 nM oleandrin for 24 h, LiCl + oleandrin (time) and LiCl + oleandrin (concentration). For the LiCl groups, the cells were pretreated with 20 mM LiCl for 6 h to activate the Wnt signaling pathway. After the cells were lysed with PLB for 15 min, firefly luciferase reagent LARII (100 μl) and Stop & Glo Reagent (100 μl) (Promega Corp., Madison, WI) were added. The OD values of the TOP flash and the FOP flash were detected and the TOP/FOP ratio reflected the activity of the Wnt/β-catenin signaling pathway.

Ma Y., Zhu B., Liu X., Yu H., Yong L., Liu X., Shao J, & Liu Z. (2015). Inhibition of oleandrin on the proliferation show and invasion of osteosarcoma cells in vitro by suppressing Wnt/β-catenin signaling pathway. Journal of Experimental & Clinical Cancer Research : CR, 34, 115.

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Investigating miR-145 and PCGEM1 in Prostate Cancer

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The experimental culture groups included 1) untransfected LNCaP and RWPE-1 cells (control groups), 2) cells transfected with pmiR-145 or miR-145 mimics (1.6 μg/ml and 50 nM, respectively), 3) cells transfected with the scrambled nucleotide sequence and empty vector (negative control or NC groups, 50 nM), 4) cells transfected with a miRNA inhibitor (NI group, 100 nM), 5) a negative control for NI (NCI group, 50 nM), 6) cells transfected with siRNA PCGEM1 sequence (siRNA PCGEM1 group, 50 nM). Cells in log phase growth were seeded on 6-well culture plates (2 × 10⁵ cells/well) and transfected when the cell fusion rate reached 70%. The DNA Lipofectamine 2000 or RNA Lipofectamine 2000 compound was added according to the manufacturer’s instructions (Invitrogen). After 6 h, the transfection medium was discarded. Cells were washed with serum-free RPMI 1640 and then cultured in RPMI 1640 supplemented with 10% FBS.

He J.H., Zhang J.Z., Han Z.P., Wang L., Lv Y.B, & Li Y.G. (2014). Reciprocal regulation of PCGEM1 and miR-145 promote proliferation of LNCaP prostate cancer cells. Journal of Experimental & Clinical Cancer Research : CR, 33(1), 72.

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Neuronal Transfection Efficiency Analysis

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Cortical neurons differentiated for 6–8 DIV as described above were transferred to fresh Neurobasal medium lacking antibiotics, and transfected with DNA/Lipofectamine 2000 (Invitrogen) following the indications of the manufacturer (1 μg DNA/1 μl Lipofectamine 2000). pSG5 Large T, pSG5 Large T K1, or pcDNA6/V5-His/LacZ DNA was always combined with pRFPRNAiC or pEGFPN1 DNA at a 19:1 ratio. After incubating the neurons for 2 h at 37 °C, transfection medium was exchanged by fresh Neurobasal medium containing BrdU (5 µg/ml). Neurons were then maintained in culture for 1 to 7 days. BrdU incorporation levels in response to TAg expression were identical regardless of the differentiation time (6–8 days).

Barrio-Alonso E., Hernández-Vivanco A., Walton C.C., Perea G, & Frade J.M. (2018). Cell cycle reentry triggers hyperploidization and synaptic dysfunction followed by delayed cell death in differentiated cortical neurons. Scientific Reports, 8, 14316.

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