Systematic screening of commonly used commercial transfection. See page 2 to view a typical dna transfection procedure. The intracellular trafficking mechanism of lipofectamine. Cellspecific transfection protocols thermo fisher scientific us. Add 100l of optimum to the eppendorf tube to dilute the dna and mix by tapping. Invitrogen lipofectamine 2000 transfection reagent fisher. Lipofectamine 2000 cd reagent is a proprietary animal originfree formulation for transfecting nucleic acids into eukaryotic cells. Lipofectamine plasmid transfection bridges lab protocols. Lipofectamine 3000 transfection reagent leverages our most advanced lipid nanoparticle technology to provide superior transfection performance with improved application outcomes and reproducible results. Plate an amount that will result in approximately 85 to 90% confluence see table below on the day of transfection.
Lipofectamine 2000 is commonly used for sirna transfections. If you still have this tox effect, then you should think about testing polymerbased reagents. The weight ratio of lipofectamine 2000 to pdna was kept constant at 3. Does anyone have experience with the lipofectamine 2000 toxicity. Lipofectamine 2000 cd reagent complexes can be added directly to cells in culture medium.
You can find in the instruction manuals of these two reagents a simple way of doing so. I follow the standard recommended testing procedures and protocol for transfection. 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. Herein, we provide data of a comparative study on optimization of transfection condition by electroporation and chemical methods for huh7 and vero cells. The impact of transfection mediated toxicity mirus bio. Yous uggested to thaw a new vial of cells if the cells had reached confluency even if it was few passages before.
Lipofectamine is formulated to give high transfection efficiency with little toxicity to cells. For example, liver and kidneyderived cell lines may be more suitable for toxicity testing. Lipofectamine 2000 is a proprietary formulation for the transfection of nucleic. Cho k1, hamster ovary, plasmid dna, lipofectamine ltx, 15338100 15338500 pdf. What percentage confluency should be used for stable transfection. In tube b place 190 l of optimemi and 10 l lipofectamine 2000. Make sufficient 10% fbs in optimem media wo antibiotics sterile filter 2. Clinical laboratory products microsatellite instability testing. Comparison of lipofectmax and lipofectamine 2000 in plasmid transfection on mouse neuronal cells.
Transient plasmid dna transfection into hela and hacat cells. Superior performance for co transfection of sirna and plasmid dna. When a finite cell line undergoes transformation and acquires the ability to. Cho k1, hamster ovary, plasmid dna, lipofectamine ltx, 15338100 15338 500 pdf. Exceptional transfection efficiency in the broadest range of cell lines and the highest levels of recombinant protein expression. However, few studies have examined cellular responses to this delivery system. Reverse transfection protocol for dna in 96well plates using transitx2 dynamic delivery system. 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. One day before transfection, plate cells in growth medium without antibiotics so that they will be 9095% confluent at the time of transfection 0. I am about to start my stable transfections with lipofectamine 2000. Lipofectamine 2000 transfection for hek293 cells about 6070% transfection rate transfect cells 6well plate. Transfection of nih3t3 cells, hela, swis 3t3, 293t with. A sample protocol is listed here for transfection experiments performed in 6well plates. Lipofectamine 2000 reagent protocol 202lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart.
Effects of lipofectamine 2000sirna complexes on autophagy in. 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. Lipofectamine 2000 reagent thermo fisher scientific. Optimising c2c12 muscle myoblast transfection with. 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.
Does it mean that once hek293 cells reached 100% confluency, it cannot be used at all for transfection expts. It is used to increase the transfection efficiency of rna including mrna and sirna or plasmid dna into in vitro cell cultures by lipofection. To perform transfection experiments in other cell culture plates, simply multiply the suggested quantities by the relative surface area of your plate. Studies were undertaken to evaluate and compare the transfection efficacy.
Invitrogen lipofectamine 3000 transfection reagent. Transfection protocol of lipofectamine rnaimax can be obtained from the invitrogen website. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. Highest transfection efficiency in many cell types and formats e. Lipofectamine 2000 transfection reagent is versatile transfection reagent that has been shown to effectively transfect widest variety of adherent and suspension cell lines. Troubleshooting for transfection of the crisprcas9 using. The mechanism of lipofectamine 2000 mediated transmembrane. The amount of lipofectamine 2000 reagent required for successful transfection varies depending on the cell type and passage number. General procedure for transfection of mammalian cells with. Prewarm 50ml of optimum stored in cold room at 4c 10min. On the reliability and validity of manual muscle testing. 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. Researchers use lipofectamine 2000 reagent for sirna and shrnabased gene knockdown experiments, as well as for gene expression studies.
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. Lipofectamine 2000 dna transfection reagent protocol. I wondering how much dna i should use for stable transfection with lipofectamine 2000 tranfection reagent. Lipofectamine 2000 transfection protocol 10cm plates reagents. C2c12, mouse muscle myoblast, plasmid dna, lipofectamine 3000. Viafect transfection reagent is a highperforming, lowtoxicity reagent. Lipofectamine 2000 reagent protocol 202 lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. Start any new transfection by testing the recommended four concentrations of lipofectamine 2000 reagent to determine an optimum amount. I have used both lipofectamine 2000 and 3000 and always get the same results.
Invitrogen lipofectamine 2000 transfection reagent. Specially designed cationic lipids, such as lipofectamine transfection reagents, facilitate. Transient transfection of mammalian cells with lipofectamine. Both lipofectamine 2000 and 3000 work well for 293t cells, you can just follow their protocol. Plate cells for 212hrs but not over 24 hours prior to transfection to achieve a 4080% confluence at the time of transfection. Mouse neuronal cells were transfected with a gfpexpressing plasmid using either lipofectmax transfection reagent left, or lipofectamine 2000 right. I transfect my pc3 cell line with lipofectamine 2000 and my sirna plk1 and with the. Lipofectamine 2000 transfection reagent from thermo fisher. For additional information on scaling your transfection reaction, see page 4. 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. Optimisation of dna transfection of the c2c12 muscle myoblast cell line, comparing use of polyethylenimine pei and lipofectamine2000. Do any one has protocol for stable transfection with lipofectamine 2000.
I am performing transfection on mcf7 cells using lipofectamine 3000 invitrogen but unable to get any transfectants, instead the cells are dying after incubation with reagent and dna. Viafect transfection reagent, lipofectamine 2000 transfection reagent or lipofectamine 3000 transfection reagent following manufacturer instructions, applying either 5l. Does anyone have experience with the lipofectamine 2000. How does transfection with lipofectamine 2000 work. Lipofectamine 2000 reagent delivers dna or sirna with excellent transfection performance for protein expression, gene silencing, and functional assays. Invitrogen lipofectamine 2000 transfection reagent life. Nov 20, 2008 i have a question regarding confluency of hek293 cells for transfection. Viafect transfection reagent transfection assay e4981. The intracellular trafficking mechanism of lipofectaminebased transfection reagents and its implication for gene delivery. 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. The transfection efficacy of lipofectamine 2000 was compromised by its high toxicity.
I use 24well plates for transfections in triplicates. Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations. Theres a difference between the two protocols lipofectamine 3000 has an extra reagent that needs to be added. Transfection efficiencies were assessed by gfp signal. Lipofectamine transfection of c2c12 cells bridges lab. Genscript recommends using lipofectamine 2000 for all transfections. May 11, 2016 the intracellular trafficking mechanism of lipofectaminebased transfection reagents and its implication for gene delivery. Lipofectamine 2000 transfection reagent is a proprietary formulation for transfecting. 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. In tube a place 2 g of dna in optimemi for a final volume of 200 l. Invitrogen lipofectamine 3000 transfection reagent fisher. Sep, 2005 invitrogens lipofectamine 2000 is one of many cationic lipidbased transfection reagents presently on the market. 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 reagent provides high transfection efficiency in many cells types and formats. The purpose of this study is to evaluate the effect of sirna transfection using lipofectamine 2000 on cellular autophagy. However, for cotransfection of sirnachimera and plasmid dna, lipofectamine 2000 should be used. Lipofectamine 3000 transfection reagent kit youtube. Invitrogen lipofectamine transfection reagent protocols have been. Add 6l of lipofectamine2000 from biostores and 100 l of. Lipofectamine 2000 transfection reagent is best transfection reagent available from life technologies for the co transfection of sirna and plasmid dna. But muscle, for example, will be hard to transfect. May 11, 2016 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, intracellular trafficking, endosomal escape and nuclear entry 1,2,3,4,5,6,7,8. Lipofectamine or lipofectamine 2000 is a common transfection reagent, produced and sold by invitrogen, used in molecular and cellular biology. 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.