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HUH7 and HEP3B cells were transfected with MKK6 S207E,T211E, p38 gamma, and p38 gamma M109T plasmids following the Lipofectamine 3000 protocol (Invitrogen). For growth inhibition assays, 5 × 10 4 cells were plated per well in a 96-well dish with 0.2 μg DNA per well.
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Important tips for optimization of transformation process: Confirm that the cells are still frozen and dry ice is still present upon receipt. For maximum transformation efficiency, use high quality DNA sample free of phenol, ethanol, proteins, salts or detergents.
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Aug 20, 2015 · Lipofectamine 3000 and RNAiMAX outperformed Lipofectamine 2000 in HEK 293 cells (Figure S1), which is in agreement with the recent finding that RNAiMAX performed better than Lipofectamine 2000 for delivery of Cas9 RNPs with low cell toxicity (Zuris et al., 2014).
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Lipofectamine 3000™ was the most potent vector compared to polyethylenimine (PEI) and Turbofect. When co-cultured with astrocytes, the genetically modified RBECs secreted recombinant EPO into the cell culture medium both luminally and abluminally, and despite lower levels of EPO reaching the abluminal chamber, the amount of recombinant EPO ...
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Optimizing Transfection To obtain the highest transfection efficiency and low non-specific effects, optimize transfection conditions by varying cell density and Lipofectamine 2000 CD concentrations.Lipofectamine 3000 reagent provided higher GFP transfection efficiency than Lipofectamine 2000 and FuGENE HD reagents for all five cell lines. Figure 2. The transfection protocol for Lipofectamine 3000 was developed to be easy to use while still ensuring optimum performance and reliability in a wide panel of cell lines.
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Lipofectamine 3000 reagent maintains a high transfection efficiency within a robust dynamic range of lipid doses for quick and easy optimization. A low toxicity lipid dose (0.75µL for 24-well plates) is suggested for applications requiring minimal disruption of the cells. The aforementioned miR-21-targeting C-HCR system was transfected into MCF-7 cells via lipofectamine 3000, and incubated with cells at 37 °C for 2 h. An intense FRET signal was observed, indicating a high miR-21 expression level in live MCF-7 cells, and the C-HCR platform offered distinct localization signals in each cell (sample a of Fig. 6(A ...
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Lipofectamine® RNAiMAX Transfection Reagent provides the highest transfection efficiencies on the widest variety of cell types for siRNA-mediated gene knockdown experiments. Lipofectamine® RNAiMAX is a proprietary RNAi-specific cationic lipid formulation designed specifically for the delivery of siRNA and miRNA into all cell types. Dec 12, 2014 · Optionally, transfection complexes of the present invention may include commercially available transfection reagents containing cationic lipids such as Lipofectin®, LIPOFECTAMINE™ RNAiMAX and LIPOFECTAMINE™2000, LIPOFECTAMINE® 3000, LIPOFECTAMINE® LTX (Life Technologies Corporation, Carlsbad, Calif.).
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2.1.1. Optimization of SSLNPs for siRNA Delivery . Lipid-Z (detail on the lipid synthesis is provided under experimental section) was prepared, purified and characterized by reversed-phase high-performance liquid chromatography (RP-HPLC) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) ().
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Results for the optimization of lipofection conditions for HMC-1.2 cells using Lipofectamine 3000 are available in Supplementary TableS1.Thephysicalproperties(size,polydispersity,andDNAencap-sulation efficiency) of the various plasmid-containing LNP formula-tions tested in this study are available in Supplementary Table S2. Dec 20, 2016 · Single-cell suspensions are seeded on glass bottom dishes at 75% confluence and allowed to grow for 24 h for transfection with plasmids using Lipofectamine 3000. On the day of transfection, medium of cells is changed to Opti-MEM (1 ml) in the absence of serum and antibiotics and transfection performed as described in Appendix I .
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Lipofectamine TM2000 according to the manufacturer’s was obtained from Invitrogen Corporation (Carlsbad, USA). ... centrifuged at 3000 rpm for 30 min to remove ... Figure 1: Example transfection workflow using the Lipofectamine® 3000 transfection reagent. Considerations for electroporation Electroporation is mainly dependent on the combination of three electric parameters: the pulse voltage, pulse width, and pulse number.
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Lipofectamine 3000 reagent was purchased from Invitrogen (Carlsbad, CA, USA). The extracellular signal‐regulated kinase (ERK) inhibitor U0126, phospho‐ERK antibody, total ERK antibody and peroxidase‐labeled secondary antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA).
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Optimization Protocol for DNA Transfection. ... transfection efficiencies with TransIT®-2020 and TransIT®-LT1 were clearly higher than with Lipofectamine® 3000. Lipofectamine® 3000 Transfection Reagent leverages our most advanced lipid nanoparticle technology to provide superior transfection performance with improved application outcomes and reproducible results.
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for optimization of transfection and in vivo efficiency is critical to ... 40 nM with Lipofectamine concentrations at 1.5 µl/well. The ... 3,000 2,000 1,000 0 GDF-11-3-UTR-WT, GDF-11-3-UTR-Mut, ROCK2-3-UTR-WT, or ROCK2-3-UTR-Mut vectors were cotransfected with miR-373 mimic or negative control into 293FT cells using Lipofectamine 3000 for 48 h. Transfected cells were analyzed using the dual-luciferase reporter assay system (Promega).
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FIG. 14 shows optimization of RNA delivery. FIG. 15 demonstrates double strand breaks at target sites. The gene targeting was successful in inducing double strand breaks in T cells activated with anti-CD3 and anti-CD28 prior to introduction of the targeted CRISPR-Cas system. Sep 26, 2019 · Cells were transiently transfected using Lipofectamine 3000 (Thermo Fisher Scientific) according to the manufacturer's instructions. For the establishment of stable cell lines, the pTsin‐HA‐SPOP mutant expression and virus packing constructs were transfected into 293T cells.
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Lipofectamine 3000 reagent provided higher GFP transfection efficiency than Lipofectamine 2000 and FuGENE HD reagents for all five cell lines. Figure 2. The transfection protocol for Lipofectamine 3000 was developed to be easy to use while still ensuring optimum performance and reliability in a wide panel of cell lines. Lipofectamine 3000 transfection protocol keyword after analyzing the system lists the list of keywords related and the list of websites with related content, in addition you can see which keywords most interested customers on the this website