Invitrogen lipofectamine 3000 transfection reagent. This result shows that simple, lipidmediated transfection of hep g2 cells is a viable alternative to gene. The intracellular trafficking mechanism of lipofectaminebased. Optimum amount needed is determined from the protocol for lipofectamine 3000 transfection reagent. To optimize the amount of lipofectamine 2000 for transfection in a 24well plate, start. Lipofectamine 2000 cd reagent complexes can be added directly to cells in culture medium. Make sufficient 10% fbs in optimem media wo antibiotics sterile filter 2. The intracellular trafficking mechanism of lipofectamine. This shows that the lipofectamine 2000 concentration should be optimized for each cell type. Although lipofectamine 2000 is compatible with most cell lines, the transfection efficiency cannot be sustained in prolonged high cell density culture for large scale operation 1. At the foundation of the popularity of this broadspectrum reagent is the reliably high efficiency and simple protocol for success across a broad range of cell lines figure 1. Its supposed to be 7090% by using lipofectamine 3000, considering hela cells are easy to be transfected. This is the first time i will have used lipofectamine 3000, previously i only used lipofectamine 2000, and i am wondering if anyone has found an optimal lipo 3000 dna ratio for this cell line. For additional information on scaling your transfection reaction, see page 4.
Genscript recommends using lipofectamine 2000 for all transfections. The reagent provides high transfection efficiency in many cells types and formats. I use 24well plates for transfections in triplicates. The lenti293t is an hek 293 derived cell line stably expresses the sv40 large t antigen and neomycin resistance gene.
Lipofectamine 2000 cd reagent complexes can be added. Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations. Cells were then stained onchip with the livecell stain cbnf and analyzed on the 2100 bioanalyzer. Now my transfection efficiency is higher and more importantly cells are healthier as the % of surviving cells is much higher. A sample protocol is listed here for transfection experiments performed in 6well plates. Also, the transfection efficiency is not very high about 30 %. What is a typical transfection efficiency in % for any of you using lipid based reagents and what cell lines did you use. Transfection the delivery of dna or rna into eukaryotic cells is a powerful tool used to study and control gene expression. Transfection of nih3t3 cells, hela, swis 3t3, 293t with. The firstgeneration lipofection was a singlecomponent system, in which only the lipid nl. The day transfection per dnalipo p3000lipo 3000 reagent complex.
Transfection efficiency of p19 cells is between 3050% from 5099% for many other stable cell lines. Refer to the cell lines database for a list of cell types successfully transfected. Lipofectamine 2000 transfection reagent from thermo fisher. Introducing the monster green fluorescent protein phmgfp vector using the transfast transfection reagent and the optimized transfection conditions resulted in the cell notes issue 7 2003 14 neuroscience transfecting a human neuroblastoma cell line with monster green fluorescent protein by natalie betz, ph. Lipofectamine 2000 transfection reagent is a proprietary formulation for the transfection of nucleic acids dna and rna into eukaryotic cells and provides the following advantages. Lipofectamine 2000 transfection, the expression of gfp was detected in 76,4% of hela cells with a mean fluorescence intensity of 437.
Lipofectamine 2000 cd reagent is a proprietary animal originfree formulation for transfecting nucleic acids into eukaryotic cells. Lipofectamine 3000 reagent maintains a high transfection efficiency within a robust dynamic range of lipid doses for quick and easy optimization. Researchers use lipofectamine 2000 reagent for sirna and shrnabased gene knockdown experiments, as well as for gene expression studies. Transfection of mcf7 with lipofectamine 2000 maartje vogel. Cloned genes can be transfected into cells for biochemical characterization, mutational analyses, investigation of the effects of gene expression on cell growth, investigation of gene regulatory elements, and to produce a specific protein. How can i optimize the transfection efficiency using. Invitrogen lipofectamine 2000 transfection reagent. Invitrogen transfection guide available at for a list of cell lines. Lipofectamine 2000 reagent delivers dna or sirna with excellent transfection performance for protein expression, gene silencing, and functional assays. It is a relatively modern method first described by felgner et al. Laboratory 2010 lipofectamine 2000 transfection protocol 10cm plates reagents. It is widely accepted that the final transfection efficiency te of lipidbased.
Lipofectamine 2000 is a cationic liposome based reagent that provides high transfection efficiency and high levels of transgene expression in a range of. Monitoring transfection efficiency in cells using an on. Lipofectaminez 2000 is a proprietary formulation suitable for the transfection of nucleic acids into eukaryotic cells. Transient plasmid dna transfection into hela and hacat. Clathrin and caveolaemediated endocytic pathway contributions to transfection efficiency were studied. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines.
In hacat cells, lipofectamine 2000 achieved a transfection efficiency of 22. The peyfpnuc plasmid dna was used to monitor transfection efficiency and as a bacterial cloning vector for mock dna transfection. Transitpro employs both mecha nisms of action and works by forming lipopolyplexes. After incubation, remove media and replace with fresh media. Plate cells so they will be 6080% confluent at the time of transfection. I want to try to use hela cell and lipofectamine 2000 to. It is best to use polypropylene instead of polystyrene tubes. Lipofectamine 2000 please follow suggested guidelines for 150mm plates. It consistently produces high transfection efficiency and high protein overexpression. Transfection protocol lipofectamine 3000 transfection. For me, fugene and lipofectamine 3000 not 2000 worked the best, while the worst for me were pei and metafectene.
Original article an improved method for increasing the efficiency of. Highest transfection efficiency in many cell types and formats e. Transfection was measured by luciferase activity using a conventional assay. Lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. Add the lipofectaminedna mixture to each well and gently swirl to mix. Transfecting a human neuroblastoma cell line with monster. We have also used lipofectamine 2000 to introduce sirna into primary synoviocytes and obtained a comparable transfection efficiency when transfecting larger dna species scott et. Evaluation of transfection methods for transient gene. Nucleic acidlipofectamine 2000 complexes can be added directly to cells in culture medium, in the presence or absence of serum. Transfection efficiencies lower than 50% were not reported in this table. How can i optimize the transfection efficiency using lipofectamine 3000invitrogen. Lipofectamine 2000 transfection for hek293 cells about 6070% transfection rate transfect cells 6well plate. Lipofectamine 2000 transfection protocol 10cm plates reagents. To perform transfection experiments in other cell culture plates, simply multiply the suggested quantities by the relative surface area of your plate.
Lipofectamine rnaimax transfection protocol see page 2 to view a typical rnaimax transfection procedure. Optimized conditions for successful transfection of human. Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. Lipofectamine 2000 dna transfection reagent protocol. Lipofectamine 2000 transfection reagent is a versatile transfection reagent that has been shown to effectively transfect the widest variety of adherent and suspension cell lines. Blue fluorescence histograms of cbnf positive cells and percentages of transfection are shown. The intracellular trafficking mechanism of lipofectaminebased transfection reagents and its implication for gene delivery.
Highest transfection efficiency in many cell types and formats animal originfree formulation ensures that mammalian cell culture and bioproduction processes are free of animalderived materials dnalipofectamine 2000 cd complexes can be added directly to cells in culture medium. Using lipofectaminez 2000 for transfection provides the following advantages. In biontex experiments gfp positivity increased to 90. Start any new transfection by testing the recommended four concentrations of lipofectamine 2000 reagent to determine an optimum amount. If the transfected dna contains a fluorescent protein, check for transfection efficiency by viewing in a fluorescent microscope. Invitrogen lipofectamine 2000 cd transfection reagent 1ml. In general, 3000 will have a better transfection efficiency 75% whereas 2000 may only be 4050%. Ive heard people claim some very high transfection efficiencies using lipofectamine 2000 or brand x lipid reagent. Spain 34 91 590 5200 sweden 08 555 12700 switzerland 061 717 95 55 taiwan 886 2 2578 7189 united kingdom 020 8328 2000.
Lipofectamine 2000 for transfection provides the following advantages. The amount of lipofectamine 2000 reagent required for successful transfection varies depending on the cell type and passage number. The i nhibitors of endocytosis were used to treat hep2 cells before lipofectamine 2000pgfpn2 transfection. Lipofection has become more common than the capo 4 precipitation, because it is just as simple to use but frequently provides a much better transfection efficiency. This is the first time i will have used lipofectamine 3000, previously i only used lipofectamine 2000, and. Im trying to express my proteins in nih3t3s, which ive heard are supposedly easy to transfect. Lipofectamine 2000 transfection reagent is versatile transfection reagent that has been shown to effectively transfect widest variety of adherent and suspension cell lines. The lenti293t cell line has been tested as a suitable cell line for lentiviral production due to high transfection efficiency, high level of protein yield and high titer of lentiviral production. Multipurpose transfection reagents product information. In detail, after 1 h of incubation of cells with lipoplexes, the fm463 marker was added following manufacturers instructions. The highest transfection efficiency in many cell types and formats e.
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