An improved method for increasing the efficiency of gene. Lipofectamine 2000 from invitrogen biocompare product. Sep 19, 2018 luciferase activity was measured post transfection of a luciferase coding plasmid using different chemical transfection reagents, transitx2, lipofectamine 2000, and lipofectamine 3000. Three cell densities of hepg2 cells were transfected with gapd sirna 100 nm using a range of volumes 0. Protocol outline for transfection of pluripotent stem cells with lipofectamine 3000 reagent. Dilution times or complexing incubation period was too short. Lipofectamine 2000 is a cationic liposome based reagent that provides high transfection efficiency and high levels of transgene expression in a range of mammalian cell types in vitro using a simple protocol. The prepared mix is enough to have triplicates 96well, duplicates 24well, and single well 6well transfections, and account for pipetting variations. Transfection guide overview of transfection methods promega. Component 96well 24well 6well final dna per well 50100 ng 250500 ng 1. Cells were transferred into multiwell plates from geltrex matrixcoated plates and transfected with lipofectamine 3000 reagent.
Assay samples to determine the transfection efficiency e. Nucleic acid lipofectamine 2000 complexes can be added directly to cells in culture medium, in the presence or absence of serum. Add the oligo merlipofectamine 2000 complexes to each well containing cells and medium. Transfection protocol lipofectamine 3000 transfection reagent. The animal originfree formulation ensures that mammalian cell culture and bioproduction processes are free of animalderived materials. Use a range of 20100 nm stealth rnai or sirna for 24well format. Change medium before transfection when using lipofectamine 2000. Transfection guide overview of transfection methods.
Determination of viability, metabolic activity, and transfection efficiency. Refer to the cell lines database for a list of cell types successfully transfected. Highest transfection efficiency in many cell types and formats e. Specifically optimized to provide exceptional transfection efficiency of plasmid dna in cho cells and cell types of associated lineage 2. Process for an efficient lentiviral cell transduction. To assess transfection efficiency, cells were first visualized via fluorescence microscopy for qualitative assessment of protein expression, morphology, and viability figure 1. Researchers use lipofectamine 2000 reagent for sirna and shrnabased gene knockdown experiments, as well as for gene expression studies. I want to try to use hela cell and lipofectamine 2000 to. This reagent delivers superior transfection efficiency and improved cell viability for the widest range of hardtotransfect and common.
Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations. Lipofectamine 3000 reagent provided higher gfp transfection efficiency than lipofectamine 2000 and fugene hd reagents for all five cell lines. Troubleshooting for transfection of the crisprcas9 using lipofectamine razieh monjezi. Transfection efficiencies were optimal around 40% of transfected cells per spot with 11. In animal cells, transfection is the preferred term as. Serum compatible with manual 1 siportneofx transfection agent. Transfection was measured by luciferase activity using a conventional assay. Also, the transfection efficiency is not very high about 30 %. It is best to use polypropylene instead of polystyrene tubes. Lipofectamine 2000 transfection reagent is best transfection reagent available from life technologies for the co transfection of sirna and plasmid dna. Lipofectamine 3000 reagent thermo fisher scientific. Lipofectamine messengermax reagent protocol outline a. Lipofectamine 2000, and lipofectamine 2000 cd x broad spectrum dna deliveryachieve high expression in many cell types, including hardtotransfect cells and insect cells x animal origin freetransit2020 provides high performance with maximum compatibility description transit2020 reagent is a new and versatile transfection solution for.
Invitrogen lipofectamine 2000 transfection reagent. Efficiency of gene transfection reagents in ng10815, shsy5y. Troubleshooting for transfection of the crisprcas9 using. Comparison of transfection efficiency of polymerbased and. On the day of transfection, which should be 1 day following cell plating, perform the following steps, which have been optimized for a single well of a 24well plate using lipofectamine 3000 transfection reagent. Plate cells so they will be 7090% confluent at the time of transfection.
Today i made a test, i transfected myoblast with gfp in following conditions. Biomodule transfection and control user manual unit with. Comparison of primefect tm and lipofectamine 2000 in plasmid transfection on mouse neuronal cells. Transfection efficiency was assessed using flow cytometry and fluorescence imaging. We dissect at p0, plate in neurobasal a with b27, glutamax and penstrep transfections are at 610 div, transfection medium is on neurons for 3 hours, both 0. Based on your initial results, you may need to optimize the electroporation parameters for your cell type. Lipofectmax transfection reagent is a lipidbased transfection reagent that forms a complex with dna or rna, and transports the complex into a variety of adherent and suspension cell lines. We have compared the efficiency of three different chemical transfection reagents lipofectamine 2000, lipofectamine ltx and transitlt1. Mar 27, 2007 too much lipofectamine will be too cytotoxic, and too little nucleic acid will give low efficiency of transfection. Overexpression transfection protocol drum lessons groningen. We examined the efficiency and cytotoxicity using nine different cell lines mc3t3e1 mouse preosteoblasts, pt30 human epithelial precancer c. Optimizing transfection lipofectamine 2000 cd reagent. What is nice about invitrogens lipofectamine 2000 is that it has an extensive, very useful manual which addresses these issues and helps tremendously cut down the initial time of protocol optimization.
Lipofectamine 3000 transfection reagent thermo fisher bioz. To perform transfection experiments in other cell culture plates, simply multiply the suggested quantities by the relative surface area of your plate. Lipofectamine 3000 and rnaimax outperformed lipofectamine 2000 in hek 293 cells figure s1. Here we compared the transfection and viral transduction efficiency using commercially available transfection reagents including fugene 6, lipofectamine 2000 and lipofectamine 3000 in different cell lines and primary cultured cells. Lipofectamine 2000 works best in cultures that are 90% at the time of transfection. Lipofectamine 2000 cd reagent is a proprietary animal originfree formulation for transfecting nucleic acids into eukaryotic cells.
Transfection efficiencies lower than 50% were not reported in this table. The i nhibitors of endocytosis were used to treat hep2 cells before lipofectamine 2000 pgfpn2 transfection. Component 96well 24well 6well final dna per well 100 ng 500 ng 2500 ng final lipofectamine 2000 reagent per well 0. Several cell densities and lipid volumes were investigated to determine optimal transfection conditions, shown by the shaded boxes. How can i optimize the transfection efficiency using. Optimized cationic lipidbased gene delivery reagents for use. Puro screening lentivirus yielded the highest cell transfection efficiency, which exceeded 90%, followed by roche reagent and then by lipofectamine 2000. Using lipofectamine 2000 to transfect eukaryotic cells offers the following advantages. Transfection efficiencies were assessed by gfp signal.
Refer to the manual for using the 18well or preprogrammed, 24well. Hela transfection is usually pretty standard in terms of protocols just make sure your nucleic acid concentration is conforming to that of the protocol youre. The mechanism of lipofectamine 2000 mediated transmembrane. L mrna transfection mrna can be transfected in a 24well plate with 1 or 2.
Invitrogen lipofectamine 2000 cd transfection reagent. I want to try to use hela cell and lipofectamine 2000 to do. Invitrogenlipofectamine 2000 transfection reagent 1. To obtain the highest transfection efficiency and low nonspecific effects, optimize transfection conditions as described below.
An electroporation protocol for efficient dna transfection in pc12. Troubleshooting transfection experiments thermo fisher. Mouse neuronal cells were transfected with a gfpexpressing plasmid using either primefect transfection reagent c, or lipofectamine 2000 d. Transfection amounts component 96well 24well 6well final mrna per well 100 ng 500 ng 2500 ng final lipofectamine. This study compared six commercially available reagents arrestin, expressfect, fugene hd, jetpei, lipofectamine 2000, and superfect for gene transfection. To obtain the maximum transfection efficiency and low nonspecific effects, you should perform optimization of transfection conditions by varying rna and lipofectamine 2000 concentrations. Flow cytometry and cck8 test showed that the cytotoxicity was the mildest with roche reagent, moderate with lentivirus, and severe with lipofectamine 2000. In this study, the optimal dose of lipofectamine 3000 and turbofect to transfect adherent cell lines such as chok1 and hek293 cells in comparison with nonadherent h9tcells with pegfpn1 and pcdh was identified. Lipofectamine 2000 transfection reagent from invitrogen.
Comparison of transfection efficiency of nonviral gene. Can anyone recommend a transfection reagent or method for. Can i use the lipefectamine2000 for mimic mirna transfection. Achieve 99% transfection efficiency in a single stroke. Transfection was investigated using various reagents, transfast, transfectam, lipofectamine 2000, and transitneural reagents, and initial experiments demonstrated. For adherent cells, we recommend 9095% confluence at the time of transfection for high efficiency, high. Genetic transfection an overview sciencedirect topics. Lipofectamine 2000 dna transfection reagent protocol. Im really at a loss, i should be seeing about 60% transfection efficiency for lipofectamine 3000, and 90% for electroporated cells. Highefficiency gene transfection of macrophages by lipoplexes. The main advantage of using lipofectamine 2000 however, was its ability to transfect a range of very different cell types coupled with a high efficiency of transfection.
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 dna lipofectamine 2000 cd complexes can be added directly to cells in culture medium. Cells were then stained onchip with the livecell stain cbnf and analyzed on the 2100 bioanalyzer. When working with lower cell densities transfection reagent as an alternative if cell viability is more important than expression levels. Transfection protocol lipofectamine 3000 transfection. The reagent provides high transfection efficiency in many cells types and formats. Transfection protocol of lipofectamine rnaimax can be obtained from the invitrogen website. It may also refer to other methods and cell types, although other terms are often preferred. For all transfection experiments, we plated the cells in clear 24well tissue culture plates between 312.
Clathrin and caveolaemediated endocytic pathway contributions to transfection efficiency were studied. Transfection efficiency was evaluated with green fluorescence protein gfp expression assays. Equal to or greater transfection efficiency compared with lipofectamine 2000 in sirna transfection figure 4. Comparative transfection of dna into primary and transformed. Optimum amount needed is determined from the protocol for lipofectamine 3000 transfection reagent. Lipofectamine 3000 is a new transfection reagent which is claimed to be more efficient than other transfection reagents like turbofect. Because of its high transfection efficiency figure 42,3, lipofectamine 2000 is also costeffective. Lipofectamine ltxplus works well in nih3t3 cells 65% gfp transfection efficiency, it will give you higher transfection efficiency in your mef cells. Lipofectamine 2000 reagent thermo fisher scientific.
Lipofectamine 2000 cd reagent complexes can be added directly to cells in culture medium. We did not find substantial transfection efficiency variations in. Lipofectamine 2000 transfection reagent is versatile transfection reagent that has been shown to effectively transfect widest variety of adherent and suspension cell lines. Transfection is the process of deliberately introducing naked or purified nucleic acids into eukaryotic cells.
For lipofectamine 2000, we recommend that the dna and transfection reagent be diluted separately and incubated for five minutes. Invitrogen lipofectamine 2000 transfection reagent is a proprietary. Mix gently and incubate for 20 minutes at room temperature solution may appear cloudy. Transfection efficiency and protein expression using geneart crispr nuclease vector. Feb 08, 2010 the transfection efficiency and viability with lipofectamine 2000 was 98% and 67%, respectively. In cell suspension, best results were obtained with jetprime and pei with transduction efficiencies of slightly above or below 4%, respectively. The transfection protocol for lipofectamine 3000 was developed to be easy to use while still ensuring optimum performance and. However, mixing lipofectamine 3000 and lipofectamine ltx for a combined use actually led to a substantial rise in transduction efficiency to.
If using a different transfection reagent, please consult the product manual. Gene delivery reagents lipofectamine 2000 and exgen 500 produced more effective transfection in ng10815 cells whereas lipofectamine 2000, exgen 500 and turbofectin 8. Advanced transfection with lipofectamine 2000 reagent. Lipofectamine transfection of c2c12 cells bridges lab. Transfection optimizationhow to get it right biocompare. For some cell types, such as cos7, the transfection efficiency was over 95% while other hardertotransfect cells, such as caco2, displayed efficiencies around 75%. We did not find substantial transfection efficiency variations in different batches of lipofectamine 2000. To optimize the amount of lipofectamine 2000 for transfection in a 24well plate, start. When you use lipofectamine 2000, youll use less transfection reagent per reaction than you would with other transfection reagents figure 5, resulting in significant savings. Transfection efficiency and cytotoxicity of each reagent was identified by using flow cytometry and xtt assay, respectively. The lipofectamine 2000 and the dna are diluted in separate polypropylene tubes with a small volume of serum free media. Transfecting a human neuroblastoma cell line with monster.
Using hela cells, we have achieved 90% transfection efficiency using lipofectamine rnaimax reagent thermo fisher, silentfect biorad, oligofectamine thermo fisher, lipofectamine 2000 thermo fisher, xtremegene roche, hiperfect qiagen, and transittko mirus reagents. Transfection efficiency of p19 cells is between 3050% from 5099% for many other stable cell lines. Lipofectmax transfection reagent has been tested to work the same efficiency as lipofectamine 2000 reagent, and used for the transfection of both. For additional information on scaling your transfection reaction, see page 4. Genscript recommends using lipofectamine 2000 for all transfections. To date, how to reach the maximum transfection and viral transduction efficiency is still challenging. Toxicity was measured through the release of a cytoplasmic protein, lactate dehydrogenase ldh, into the supernatant. Blue fluorescence histograms of cbnf positive cells and percentages of transfection are shown. Lipofectamine 3000 reagent thermo fisher scientific us.
The transfection efficiency with lipofectamine plus was 82% with a viability of 80% data not shown. Lipofectamine 2000 is primarily used for delivery of plasmid dna, but it also works well for delivery of sirna and mirna mimics and of course codelivery. Transfection conditions can be easily established for automated or robotic systems used in such applications. We have used cationic lipidbased transfection reagents for ectopic gene expression experiments in developing vertebrate embryos. Transfection efficiency analysis at 48 hr following transfection of a gfp reporter construct, cells were evaluated via microscopy and flow cytometry. Rapid and highly efficient mammalian cell engineering via. Highest transfection efficiency in many cell types and formats. Htm cells were transfected using lipofectamine with a fluorescent mirna dy547 and analyzed 48 hours after transfection. Invitrogen lipofectamine 2000 transfection reagent is a proprietary formulation for the transfection of nucleic acids dna and rna into eukaryotic cells providing the following advantages. Aug 20, 2015 the commonlyused plasmid dna or rna lipofectamine transfection reagent had been shown to be able to deliver many proteins into cells, so we tested several lipofectamine variants for their ability to efficiently deliver cas9 rnps sells et al. Sep 25, 2000 the lipofectamine formulation showed a 1012fold increase in transfection efficiency over dotap and a 45fold increase over lipofectin. Dna transfection see page 2 to view a typical dna transfection procedure.
Lipofectamine, lipofectamine 2000, and lipofectamine enhanced with a disulfide linked pegylated lipid mpegssdope were initially tested and optimized in cell culture. In both these cell lines, transfection efficiency reached 6080%. However, for cotransfection of sirnachimera and plasmid dna, lipofectamine 2000 should be used. It consistently produces high transfection efficiency and high protein overexpression. Lipofectamine 2000 reagent is a lipidderived reagent designed to deliver exogenic nucleic acids dna andor rna into eukaryotic cells. I used lipofectamine 2000 reagent for transfection but the efficiency were very low re. Lipofectamine 2000 dna transfection reagent protocol see page 2 to view a typical dna transfection procedure. Lipofectamine 2000 cd reagent thermo fisher scientific. The media used for complexation can affect the efficiency of transfection, but its usually pretty minimal 510%.
Has anyone experienced very low transfection efficiency. Its supposed to be 7090% by using lipofectamine 3000, considering hela cells are easy to be transfected. In the first experiment, cell viabilitytoxicity and transfection efficiencies were determined for dnain and lipofectamine 2000 in parallel. Incubate the cells at 37c in a co 2 incubator for 2496 hours until you are ready to. Monitoring transfection efficiency in cells using an onchip. Lipofectamine 3000 was more efficient in transfecting pcdh, while turbofect was more efficient in separate transfection of chok1 and hek293 with pegfpn1. If you own this web property, please visit our service catalog page to select a preference for handling your data otherwise you may continue to the site. The one factor i would consider for your complex media, if using optimem, is the. Comparison of efficiency and cytotoxicity of different. How to increase the transfection efficiency in primary.
Lipofectamine 2000 reagent protocol 202 lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. This site is scheduled to expire on march 18, 2021 dartmouth. This transfection method showed minimal toxicity at the concentrations tested and was at least 2025fold superior to the most frequently used deaedextran method for macrophage transfection. Provides the highest transfection efficiency in a wide variety of mammalian cell types oligomer lipofectamine 2000 complexes can be added directly to cells in culture medium in the presence of serum. After 5 minutes incubation, combine the diluted dna with the diluted lipofectamine 2000 total volume is 500. Exceptional transfection efficiency in the broadest range of cell lines and the highest levels of recombinant protein expression.
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