Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. Clathrin and caveolaemediated endocytic pathway contributions to transfection efficiency were studied. Therefore, all three transfection reagents appear suitable for use in bmdm. Lipofectamine 2000 transfection reagent is versatile transfection reagent that has been shown to effectively transfect widest variety of adherent and suspension cell lines. Transfect plasmid dna and sirna at the same time using lipofectamine 2000 reagent by adding 30 pmol 0. In addition, invitrogen claims that lipofectamine 2000 gives the most proven results in more functional and phenotypic assays than any other reagent and they back up this claim with an abundance of references. Exceptional transfection efficiency in the broadest range of cell lines and the highest levels of recombinant protein expression. Transfection efficiency in a cancer cell line panel. However, for cotransfection of sirnachimera and plasmid dna, lipofectamine 2000 should be used. A guide to transient expression of membrane proteins in. Lipofectamine 2000 procedure for simultaneously transfecting. It is used to increase the transfection efficiency of rna including mrna and.
Comparison of small interfering rna sirna delivery into. Not recommended for use with lipofectamine 2000 or lipofectamine rnaimax. 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. Thermo scientific nunc 24well cellculture treated multidishes 142475 invitrogen lipofectamine 3000 transfection reagent l3000008 gibco optimem i reduced serum medium 31985062 step tube complexation components amount per well 24well. Transfection protocol of lipofectamine rnaimax can be obtained from the invitrogen website. Invitrogen lipofectamine 3000 transfection reagent fisher.
Cells should be 90% confluent at the time of transfection. 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. The most efficient reagents were the cationic lipid reagents, lipofectamine 2000 and ltx, from invitrogen, which were able to genemodify up to 38% of huvec. Lipofectamine 2000 transfection reagent is the number one choice for cotransfection, given its effectiveness for transfecting both sirna and plasmid dna. The the reagent provides high transfection efficiency in. For the cell lines easy to transfection, you can use either of the procedures reverse or forward. Lipofectamine 2000 transfection reagent life technologies, cat. Rnaimax transfection reagent 778030, invitrogen, waltham, ma. Life technologies corporation andor its affiliates warrant their. Mix optimem with dna in an eppendorf 510 g dna0,8 ml optimem 2. Pdf optimization of sw480 colon cancer cells transfection with. Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations. Contact life technologies technical service or your local sales representative for permission to crossreference the dmf.
The cells were lysed 24 hours post transfection and luciferase activity was measured using dualluciferase reporter assay system and glomax explorer promega. Lfn 2000 transfection reagent life technologies, carlsbad, ca. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. Invitrogen lipofectamine 2000 transfection reagent 1. Invitrogen lipofectamine 2000 transfection reagent fisher. The amount of lipofectamine 2000 reagent required for successful transfection varies depending on the cell type and passage number. Prewarm 50ml of optimum stored in cold room at 4c 10min. Advanced transfection with lipofectamine 2000 reagent.
Pool dishes of cells that have been prepared for transfection. Transfection protocol transfection components component cat. Contact life technologies technical service or your. Samples were run on a 412% trisbis nupage gel invitrogen catalog no. It is used to increase the transfection efficiency of rna including mrna and sirna or plasmid dna into in vitro cell cultures by lipofection. The protocal is from thermo fisher scientific important guidelines for transfection 1. Make sufficient 10% fbs in optimem media wo antibiotics sterile filter 2. Optimizing transfection of primary human umbilical vein.
Cotransfecting plasmid dna and rnai into mammalian cells. Our study demonstrated that a small selection of commercially available chemical transfection reagents was able to transfer exogenous genes efficiently to primary human cells. Transfection of mcf7 with lipofectamine 2000 maartje vogel. The diluted dna and lipofectamine 2000 were combined and incubated at. Culture cells in serumfree media that are free of animal. The inhibitors of endocytosis were used to treat hep2 cells before lipofectamine 2000 pgfpn2. Lipofectamine 2000 reagent thermo fisher scientific. The transfection efficacy of lipofectamine 2000 was compromised by its high. Add 6l of lipofectamine2000 from biostores and 100 l of. In this paper, the relatived mechanism between lipofectamine 2000 mediated transmembrane gene delivery and endocytic pathway were investigated. The intracellular trafficking mechanism of lipofectaminebased.
Lipofectamine 2000 has been used successfully to transfect synthetic short. Photograph of expected results dna lipofectamine scaling up or down. Lipofectamine 3000 transfection reagent leverages our most advanced lipid nanoparticle technology to provide superior transfection performance with improved application outcomes and reproducible results. Plate cells so they will be 7090% confluent at the time of transfection. Superior performance for cotransfection of sirna and plasmid dna. 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. The amount of lipofectamine 2000 reagent required for successful. This commercially available transfection agent consists of positively charged lipid molecules. I personally have used this reagent to transfect cells for fluorescent microscopy, gst pull downs, and coimmunoprecipitation experiments. The mechanism of lipofectamine 2000 mediated transmembrane. Optimem 1 bottle life technologies 31985070 lipofectamine 2000 1ml vial life technologies 11668019.
Cotransfecting plasmid dna and rnai into mammalian cells using lipofectamine 2000 introduction lipofectamine 2000 reagent is a proprietary formulation that facilitates highly efficient delivery of stealth rna molecules, short interfering rna sirna or plasmid dna to mammalian cells for rnai analysis 1, 2. The weight ratio of lipofectamine 2000 to pdna was kept constant at 3. Mix optimem and lipofectamine in another eppendorf 32 l lipofectamine in 0,8 ml optimem 3. By the use of this product you accept the terms and conditions of all applicable limited use label licenses. Invitrogen lipofectamine 3000 transfection reagent lipofectamine 3000 transfection reagent leverages our most advanced lipid nanoparticle technology to provide superior transfection performance with improved application outcomes and reproducible results. Hela tranfection dual luciferase assay tissue and cell culture. Dnaplus lipid complexes must be made in serumfree medium such. Transfecting sirna using lipofectamine raiax pin liu, 2017 declaration.
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. Cells were stained for pluripotency with the ssea4 antibody, visualized by. The intracellular trafficking mechanism of lipofectamine. Stem cell transfection guide introduction stem cells continue to show immense promise for the future of regenerative medicine and personalized therapeutic treatments. I use 24well plates for transfections in triplicates. Systematic screening of commonly used commercial transfection.
We have had success using lipofectamine reagents in the past and the lipofectamine 3000 appears to be the best option. Lipofectamine 2000 reagent delivers dna or sirna with excellent transfection performance for protein expression, gene silencing, and functional assays. Start any new transfection by testing the recommended four concentrations of lipofectamine 2000 reagent to determine an optimum amount. Invitrogen lipofectamine 2000 transfection reagent strong performance in a wide range of cell lines reliableone of the mostcited dna reagents strong performancefor easytotransfect to moderately difficulttotransfect cell lines easy to usesimple protocol for easy optimization in a wide range of cells. As a matter of fact, they should be evaluated in their entirety for the development of optimized nonviral gene delivery vectors. Lipofectamine 2000 transfection reagent is easy to usesimply mix with nucleic acid and add to cell culture. Evaluation of transfection methods for transient gene. Lipofectamine 2000 from invitrogen biocompare product. Lipofectamine 2000 and lipofectamine rnaimax transfection. Invitrogen lipofectamine 2000 transfection reagent is a versatile transfection reagent that has been shown to effectively transfect a variety of payloads into the widest variety of adherent and suspension cell lines. Lipofectamine 2000 cd reagent is a proprietary animal originfree formulation for transfecting.
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. Invitrogen lipofectamine 3000 transfection reagent 1. Flick each 50 ml conical tube and let sit for 5 minutes at room temperature. Lipofectamine 2000 transfection protocol 10cm plates reagents. Amanda ooi, 1 aloysius wong, 1, 2 luke esau, 1 fouad lemtirichlieh, 1 and chris gehring 1. Highest transfection efficiency in many cell types and formats.
Dna complexes were added to wells so that transfection could occur as cells were attaching to the plate. Dec 01, 2006 invitrogens lipofectamine 2000 is highly cited in the literature, a sign of a successful reagent, so when our lab embarked upon the path to develop a successful transfection protocol, we included lipofectamine 2000 along with the other leading reagents. Lipofectamine 2000 cd chemically defined transfection reagent is a proprietary animal originfree formulation for transfecting nucleic acids into eukaryotic cells. Lipofectamine 2000 transfection reagent is best transfection reagent available from life technologies for the cotransfection of sirna and plasmid dna. I am performing transfection on mcf7 cells using lipofectamine 3000 invitrogen but unable to get any transfectants, instead. Amongst the eleven transfection reagents investigated, lipofectamine 2000, lipofectamine rnaimax and dharmafect 3 consistently gave the best results, in terms of sirna uptake and target gene knockdown with minimal cell toxicity and type i ifn response induction. Laboratory 2010 lipofectamine 2000 transfection protocol 10cm plates reagents. The density of the cell should be 8090 % confluent, and the cells should be evenly distributed and attached on the dish. Does anyone have experience with the lipofectamine 2000 toxicity. Since the discovery of induced pluripotent stem cells ipscs in 2006, researchers have. Lipofectamine 2000 transfection reagent from invitrogen. Optimem reduced serum medium, no phenol red life technologies, cat. Researchers use lipofectamine 2000 reagent for plasmid dna transfection as well as sirna and shrnabased gene knockdown.
Dna complexes were added to cells that had already been allowed to attach to the plate b. A drug master file dmf has been submitted to the fda. Researchers use lipofectamine 2000 reagent for sirna and shrnabased gene knockdown experiments, as well as for gene expression studies. Does anyone have experience with the lipofectamine 2000. The neuronal cytoskeleton, motor proteins, and organelle trafficking in the axon. Invitrogen lipofectamine 3000 transfection reagent from. Theres a difference between the two protocols lipofectamine 3000 has an extra reagent that needs to be added. Lipofectamine 2000 transfection for hek293 cells about 6070% transfection rate transfect cells 6well plate. Highest transfection efficiency in many cell types and formats e. 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. Biologie cellulaire lipofectamine 2000 et antibiotiques. Transfection of nih3t3 cells, hela, swis 3t3, 293t with. A h9 escs or b ipscs were transfected using lipofectamine 3000 reagent.
Use a range of several ul of lipofectamine and ug dna to increase viability after transfection and efficiency. Lipofectamine 2000 cd reagent thermo fisher scientific. A guide to transient expression of membrane proteins in hek293 cells for functional characterization. A new transfection reagent for effective transfection of primary cells, hardtotransfect cells and sensitive established cell lines article pdf available april 2007 with 1,082 reads. Life technologies corporation andor its affiliates warrant their products as set. Add 100l of optimum to the eppendorf tube to dilute the dna and mix by tapping. For adherent cells, we recommend 9095% confluence at the time of transfection for high efficiency, high.
Application note gene delivery solutions stem cell. The manual recommends also that a certain amount of nucleic acid, described in ug, should be used for transfection. Transfection and genome engineering fisher scientific. Using lipofectamine 2000 cd reagent for transfection provides the following advantages. Jan 24, 20 60 nm antisense inhibitor mir200a or scrambled control antisense inhibitor dharmacon was transfected into mcf7 cells using lipofectamine 2000 or lipofectamine rnai max invitrogen. Nonviral carriers including those based on synthetic. Lipofectamine or lipofectamine 2000 is a common transfection reagent, produced and sold by invitrogen, used in molecular and cellular biology. Lipofectamine 2000 reagent protocol 202lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart.
Invitrogen lipofectamine 3000 transfection reagent. The intracellular trafficking mechanism of lipofectamine based transfection reagents and its implication for gene delivery. Cells were harvested with trizol reagent invitrogen. Lipofectamine 2000 cd reagent is a proprietary animal originfree formulation for transfecting nucleic acids into eukaryotic cells. This product is covered by one or more limited use label licenses see the invitrogen catalog or our website. 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. Invitrogen lipofectamine 2000 transfection reagent. Optimizing transfection lipofectamine 2000 cd reagent. Lipofectamine 2000 reagent and lipofectamine 3000 reagent were used to deliver invitrogen geneart precision tals and crispr nucleases targeting the aavs1 safe harbor locus in u2os and hepg2 cell lines in 12well format. The density of the cell should be 8090% confluent, and the cells should be evenly distributed and attached on the dish. This study shows that lipofectamine 2000 is an efficient reagent for the.
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