The impact of transfection mediated toxicity mirus bio. 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. Genscript recommends using lipofectamine 2000 for all transfections. See page 2 to view a typical dna transfection procedure. Invitrogen lipofectamine 2000 transfection reagent. The intracellular trafficking mechanism of lipofectamine. Add 6l of lipofectamine2000 from biostores and 100 l of. Trachea smooth muscle, muscle, cow, lipofectamine reagent. To perform transfection experiments in other cell culture plates.
Invitrogen lipofectamine transfection reagent protocols have been. Protocol 1 stable transfection using lipofectamine 2000 in 6 well plates. Start any new transfection by testing the recommended four. Lipofectamine 2000 transfection reagent is a versatile transfection reagent that has been shown to effectively transfect the widest variety of adherent and suspension cell. Research diagnostic development diagnostic testing precision medicine preclinical to companion diagnostic development public health. Effects of lipofectamine 2000sirna complexes on autophagy in. Optimemin ul, equal volume for dna and lipofectamine lipofectaminein ul 25. Lipofectamine 2000 transfection reagent from thermo fisher. Lipofectamine is formulated to give high transfection. The impact of transfection mediated toxicity gene expression and cytotoxicity analysis of transfection reagents introduction while plasmid dna delivery is a widely used method to. Invitrogen lipofectamine 2000 transfection reagent is a proprietary formulation for the transfection of nucleic acids dna and rna into eukaryotic cells providing the following. Advanced transfection with lipofectamine 2000 reagent. From the above data in table 3, this indicates that the 1. I have used both lipofectamine 2000 and 3000 and always get the same results.
Add 100l of optimum to the eppendorf tube to dilute the dna and mix by tapping. Viafect transfection reagent is a highperforming, lowtoxicity reagent. Enhanced transfection of cryomanipulated lf2000 is not dependent on a specific promoter. The trusted, simple, broadspectrum transfection reagent for most cell lines. Make sufficient 10% fbs in optimem media wo antibiotics sterile filter 2. Laboratory 2010 lipofectamine 2000 transfection protocol 10cm plates reagents.
Lipofectamine 3000 transfection reagent kit youtube. Lipofectamine 2000 reagent delivers dna or sirna with excellent transfection performance for protein expression, gene silencing, and functional assays. Graphpad prism software and students ttest was used to. I use 24well plates for transfections in triplicates. Transient transfection of mammalian cells with lipofectamine. C2c12, mouse muscle myoblast, plasmid dna, lipofectamine 3000. Original article an improved method for increasing the efficiency of. Comparison of three different transfection methods biology.
Invitrogen lipofectamine 2000 transfection reagent fisher. The transfection efficacy of lipofectamine 2000 was compromised by its high toxicity. Different cell confluencies, dnareagent ratios and total transfection volumes were optimized for two chemical reagents including jetpei and lipofectamine 2000. Lipofectamine 2000 transfection for hek293 cells about 6070% transfection rate transfect cells 6well plate. The delivery of dna into human cells has been the basis of advances in the understanding of gene function and the development of genetic therapies. Lipofectamine 2000 cd reagent is a proprietary animal originfree formulation for transfecting nucleic acids into eukaryotic cells. The intracellular trafficking mechanism of lipofectaminebased transfection reagents and its implication for gene delivery. The lipofectamine 3000 is the bees knees though, i managed to get loads of transfects from a difficult to transfect cell line that had been giving us trouble with the lipofectamine 2000. A sample protocol is listed here for transfection experiments performed in 6well plates. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. I want to try to use hela cell and lipofectamine 2000 to do transfection.
What percentage confluency should be used for stable transfection. Lipofectamine 2000 transfection reagent is versatile transfection reagent that has been shown to effectively transfect widest variety of adherent and suspension cell lines. Studies were undertaken to evaluate and compare the transfection efficacy. Mcf7, breast cancer, human, lipofectamine 2000 reagent. Lipofectamine plasmid transfection bridges lab protocols. Lipofectamine 2000 is commonly used for sirna transfections. However, few studies have examined cellular responses to this delivery system. One day before transfection, plate cells in growth medium without antibiotics so that they will be 9095% confluent at the time of transfection 0. Plate cells for 212hrs but not over 24 hours prior to transfection to achieve a 4080% confluence at the time of transfection. I want to try to use hela cell and lipofectamine 2000 to. Lipofectamine 2000 cd reagent complexes can be added directly to cells in culture medium. I wondering how much dna i should use for stable transfection with lipofectamine 2000 tranfection reagent. Transfection commonly refers to the introduction of nucleic acids into eukaryotic cells, or more specifically.
Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations. Exceptional transfection efficiency in the broadest range of cell lines and the highest levels of recombinant protein expression. Lipofectamine 2000 reagent thermo fisher scientific. Lipofectamine 2000 is a proprietary formulation for the transfection of nucleic. Lipofectamine reagents are widely accepted as goldstandard for the safe delivery of. Highest transfection efficiency in many cell types and formats e. Transfection protocol of lipofectamine rnaimax can be. The vector contained an ofp reporter gene and was transfected with lipofectamine 2000 or lipofectamine 3000 reagent into a u2os and b hepg2 cell lines. But muscle, for example, will be hard to transfect. I transfect my pc3 cell line with lipofectamine 2000 and my sirna plk1. Prewarm 50ml of optimum stored in cold room at 4c 10min.
The lenti293t cell line has been tested as a suitable cell line for. Plate cells for 212hrs but not over 24 hours prior to. Lipofectamine 2000 dna transfection reagent protocol. To test this hypothesis, we evaluated the te of lfn and dd on both. The amount of lipofectamine 2000 reagent required for successful transfection varies depending on the cell type and passage number. 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. Lipidbased transfection reagents exhibit cryoinduced increase in.
The transfection protocol for lipofectamine 3000 reagent was developed to be easy to use while still ensuring optimum performance and reliability in a wide panel of cell lines. The lenti293t is an hek 293 derived cell line stably expresses the sv40 large t antigen and neomycin resistance gene. Combine the two volumes of optimemdna and optimemlipofectamine. Each reagent was used to transfect hek 293, hela, lncap, a549, and hepg2 cells in 96well format. May 11, 2016 the intracellular trafficking mechanism of lipofectaminebased transfection reagents and its implication for gene delivery. Does anyone have experience with the lipofectamine 2000. How does transfection with lipofectamine 2000 work. Viafect transfection reagent transfection assay e4981. As always, use good cell culture practices and wear your personal protective. The manual recommends days whereas some people transfect for a 4. In tube b place 190 l of optimemi and 10 l lipofectamine 2000. The lipofectamine 3000 is the bees knees though, i managed to get loads.
Reverse transfection protocol for dna in 96well plates using transitx2 dynamic delivery system. The impact of transfection mediated toxicity gene expression and cytotoxicity analysis of transfection reagents introduction while plasmid dna delivery is a widely used method to study cellular functions of proteins of interest, studies to identify nontoxic gene delivery reagents are limited. 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 do. I personally have used this reagent to transfect cells for fluorescent microscopy, gst pull downs, and coimmunoprecipitation experiments. Invitrogens lipofectamine 2000 is one of many cationic lipidbased transfection reagents presently on the market. I am getting shitty transfection efficiency using effectene and geneporter 2 transfection reagent. Lipofectamine 2000 reagent protocol 202lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. The reagent provides high transfection efficiency in many cells types and formats.
To obtain the highest transfection efficiency and low nonspecific effects, optimize transfection conditions by varying rna and lipofectamine 2000 concentrations. Invitrogen lipofectamine 2000 transfection reagent 15ml. Systematic screening of commonly used commercial transfection. Plate an amount that will result in approximately 85 to 90% confluence see table below on the day of transfection. Lipofectamine is formulated to give high transfection efficiency with little toxicity to cells. Can anyone please explain why we need the p3000 in addition to lipofectamine or how p3000 helps in adition to the lipo. Troubleshooting for transfection of the crisprcas9 using. Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. Examples of cells transfected successfully thermo fisher scientific. Invitrogen lipofectamine 2000 transfection reagent life.
To perform transfection experiments in other cell culture plates, simply multiply the suggested quantities by the relative surface area of your plate. However, for cotransfection of sirnachimera and plasmid dna, lipofectamine 2000 should be used. Statistical significance was evaluated by a twotailed students ttest. I wondering how much dna i should use for stable transfection with lipofectamine 2000 tranfection. 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 transfection of c2c12 cells bridges lab.
An example of high efficiency forward and reverse transfection of a549 cells using the broad spectrum transitx2 dynamic delivery system can be found in the figure below. Cellspecific transfection protocols thermo fisher scientific us. After 4h aspirate media and refeed cells with normal media. Using cx43 shrna lentivirus combined with lipofectamine 3000 transfection. Both lipofectamine 2000 and 3000 work well for 293t cells, you can just follow their protocol.
The weight ratio of lipofectamine 2000 to pdna was kept constant at 3. Does anyone have experience with the lipofectamine 2000 toxicity. Start any new transfection by testing the recommended four concentrations of lipofectamine 2000 reagent to determine an optimum amount. It is used to increase the transfection efficiency of rna including mrna and sirna or plasmid dna into in vitro cell cultures by lipofection. Transfection of nih3t3 cells, hela, swis 3t3, 293t with. 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. You can find in the instruction manuals of these two reagents a simple way of doing so. Lipofectamine 2000 transfection reagent is best transfection reagent available from life technologies for the co transfection of sirna and plasmid dna. Transfection protocol of lipofectamine rnaimax can be obtained from the invitrogen website. Herein, we provide data of a comparative study on optimization of transfection condition by electroporation and chemical methods for huh7 and vero cells. Lipofectamine or lipofectamine 2000 is a common transfection reagent, produced and sold by invitrogen, used in molecular and cellular biology. For additional information on scaling your transfection reaction, see page 4. For more tips for your rnai experiment, refer to seven steps to rnai success. The mechanism of lipofectamine 2000 mediated transmembrane.
Ive seen it with lipofectamine 2000 or by transfection with polyethylenimine pei. Superior performance for co transfection of sirna and plasmid dna. The purpose of this study is to evaluate the effect of sirna transfection using lipofectamine 2000 on cellular autophagy. Cho k1, hamster ovary, plasmid dna, lipofectamine ltx, 15338100 15338 500 pdf. 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. It is widely accepted that the final transfection efficiency te of lipidbased transfection reagents trs is ratelimited by several biological barriers such as cellular uptake. Protocol 6 well dish plate cells and grow to 70% confluence in 2 ml media without antibiotics dmem. Lipofectamine 3000 has two main advantages over our existing reagent, the biggest one is its significantly higher transfection efficiency in difficult to transfect cell lines. Invitrogen lipofectamine 3000 transfection reagent from. Viafect transfection reagent, lipofectamine 2000 transfection reagent or lipofectamine 3000 transfection reagent following manufacturer instructions, applying either 5l.
I follow the standard recommended testing procedures and protocol for transfection. I am about to start my stable transfections with lipofectamine 2000. General procedure for transfection of mammalian cells with. The intracellular trafficking mechanism of lipofectaminebased. Sep, 2005 invitrogens lipofectamine 2000 is one of many cationic lipidbased transfection reagents presently on the market. Clinical laboratory products microsatellite instability testing. Lipofectamine 2000 transfection reagent is best transfection reagent available from life technologies for the cotransfection of sirna and plasmid dna. Help on transfecting mcf7 and hct 116 cells transfection.
From the luciferase activities and consistencies of data using different transfection reagents including genejammer, effectine, wellfect, lipofectamine, what i can say from the data i observed, the efficiency of transfection is much more better in hct116 cells using lipofectamine 2000. Theres a difference between the two protocols lipofectamine 3000 has an extra reagent that needs to be added. Dmem may be used to dilute lipofectaminez 2000, but transfection efficiency may be compromised. Add dnalipofectamine complexes 500 ul to the 2 ml of media on the cells.
Troubleshooting for transfection of the crisprcas9 using lipofectamine. Effects of lipofectamine 2000sirna complexes on autophagy. This protocol is for transfecting plasmid dna into mammalian cells. In tube a place 2 g of dna in optimemi for a final volume of 200 l. Numerous chemical and physical approaches have been used to deliver the dna, but their efficacy has been variable and is highly dependent on the cell type to be transfected. Lipofectamine 2000 transfection protocol 10cm plates reagents. We have had success using lipofectamine reagents in the past and the lipofectamine 3000 appears to be the best option. An improved method for increasing the efficiency of gene.