For more information about lipofectamine 2000 and other transfection reagents, refer to our web site or contact technical support page. Lipofectamine 2000 transfection reagent from thermo fisher. Lipofectamine 2000 and lipofectamine 3000 were used to transfect u2os and. Our linear pei protocol is as effective as commercial reagents in the transfection of hela cells and xdc293 cells, a derivative of hek293 cells, but at a fraction of the cost. The transfection protocol for lipofectamine 3000 was developed to be easy to use while still ensuring optimum performance and. Invitrogen transfection reagent, recommended transfection sku, protocol. May 11, 2016 the intracellular trafficking mechanism of lipofectaminebased transfection reagents and its implication for gene delivery. On the day of transfection, 500 ng cas9 protein and 120 ng hprt1 grna were transfected with either lipofectamine 2000 lf2k, lipofectamine 3000 lf3k, lipofectamine messengermax, lipofectamine rnaimax, turbofect or xfect transfection reagent. Luciferase expression bars and cell viability lines were measured either. Co transfection of plasmids and sirna results in reduced efficiency of plasmid delivery, but similar sirna delivery. Feb 18, 2016 to identify the best transfection reagents for delivery of cas9 rnps, initially we compared several commercially available protein transfection reagents, including lipofectamine 2000, lipofectamine 3000, lipofectamine rnaimax, lipofectamine meassengermax, turbofect, and xfect protein transfection reagent. Lipofectamine 2000 reagent protocol 202lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart.
The weight ratio of lipofectamine 2000 to pdna was kept constant at 3. Ive worked with 293t cells and i managed to always get good levels of transfection, without having to worry too much about cell density or reagent. What is the appropriate way of adding lipofectamine 3000 to cells. To perform transfection experiments in other cell culture plates, simply multiply the suggested quantities by the relative surface area of your plate. Transfection treatment effects on reference gene stability. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines.
Higher densities can lead to spontaneous differentiation. Lipofectamine 2000 reagent protocol 202 lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. The transfection protocol for lipofectamine 3000 was developed to be easy to use while still ensuring optimum performance and reliability in a wide panel of cell lines. Using lipofectamine 2000 cd reagent for transfection provides the following advantages. 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. I am trying to do transfection in mammalian cells u2os and hela with lipofectamine3000. I am actually using different cells than hela, namely rat fibroblasts and have a lot of toxicity too. Make sufficient 10% fbs in optimem media wo antibiotics sterile filter 2. Gently shake or swirl the wells or flasks to ensure even distribution over the entire plate. In a majority of cases, one wants the highest level of transfection possible with. Protocols for transfecting the most common cell lines with xtremegene transfection reagents.
Hek293 cell line transfection in 1977, human embryonic kidney cells were transformed with fragments of human adenovirus type 5, giving rise to todays widely used hek293 cell line. Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations. Plate cells so they will be 7090% confluent at the time of transfection. Well, i obtain good results using lipofectamine2000, i dont know if they work. Choosing the right transfection reagent for optimal efficiency. Lipofectamine 2000 dna transfection reagent protocol. Highest transfection efficiency in many cell types and formats e.
Transfection of nih3t3 cells, hela, swis 3t3, 293t with. U2os transient transfection posted in cell biology. I have used lf2000 successfully with the following cell lines. Lipofectamine 2000 transfection reagent is best transfection reagent available from life technologies for the co transfection of sirna and plasmid dna. Therefore, for every cell line, an appropriate transfection protocol must be established separately. Lipofectamine 2000 transfection protocol 10cm plates reagents. Lipofectamine 2000 and 3000 summary in this comparative study, transfections were performed with dnain and compared to the current market leader, lipofectamine 2000 lf2k, using a series of cultured celllines and primary cells. Hi, ive been trying to transiently transfect u2os cells to carry out coips using lipofectin lipofectamine reagents. After trying different amount of lipofectamine reagent as well as dna, it seems obvious to me too that dna and lipofectamine are really toxic. Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. Viafect transfection reagent transfection assay e4981. Improved delivery of cas9 proteingrna complexes using lipofectamine crisprmax.
A general account on neuronal cell lines and basic protocol for cell line cultures. I want to try to use hela cell and lipofectamine 2000 to do transfection. Lipofectamine 2000 transfection reagent is versatile transfection reagent that has been shown to effectively transfect widest variety of adherent and suspension cell lines. Transfection protocol of lipofectamine rnaimax can be obtained from the invitrogen website. A sample protocol is listed here for transfection experiments performed in 6well plates. 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. While lipofectamine 2000 reagent demonstrated transfection efficiencies. Lipofectamine 2000 reagent can be used for transfection of plasmid dna and small rnas. I am going to transfect plasmid in u2os cells using fugene hdviafect. Lipofectamine 3000 reagent provided higher gfp transfection efficiency than lipofectamine 2000 and fugene hd reagents for all five cell lines. Effi ciency and ofp expression were analyzed 72 hours posttransfection and a u2os and b hepg2. Lipofectamine 2000 transfection reagent dartmouth college. Sep, 2005 transfection of primary synoviocytes resulted in greater than 50% transfection efficiency. Lipofectamine 2000 cd chemically defined transfection reagent is a proprietary animal originfree formulation for transfecting nucleic acids into eukaryotic cells.
A suitable reference gene should not vary in expression between cells with and without treatments. Genscript recommends using lipofectamine 2000 for all transfections. Superior performance for co transfection of sirna and plasmid dna. A u2os cells transfected with lipofectamine 3000 reagent showed 1. While lipofectamine 2000 reagent demonstrated transfection efficiencies only slightly lower those of viafect reagent, it was considerably more toxic to hela cells at all tested ratios of reagent to dna. The intracellular trafficking mechanism of lipofectamine. A reverse transfection protocol was used to transfect mcf7 and hepg2 cells. 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. 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. In the present study, we screened a large set of transfection reagents using relatively easy and hardtotransfect cell lines and identified a new transfection reagent, lipofectamine. Optimized conditions for successful transfection of human.
The mechanism of lipofectamine 2000 mediated transmembrane. I am trying to optimize the transfection by using lipofectamine 2000 for 2000 ng of plasmid dna well with different amounts of lipofectamine. Researchers use lipofectamine 2000 reagent for sirna and shrnabased gene knockdown experiments, as well as for gene expression studies. Luciferase expression was compared at 24 hours post transfection using a standard luciferase assay. The reagent provides high transfection efficiency in many cells types and formats. Protocols for transfecting the most common cell lines with. It is possible to plate and transfect the cells on the same day, but cells must adhere to the bottom of the 6well dish. Transient plasmid dna transfection into hela and hacat cells. The fugene hd protocol database provides optimization guidelines for a. Jul 24, 2007 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. Prewarm 50ml of optimum stored in cold room at 4c 10min. Thus, we studied gene expression in the presence of lipofectamine 2000 or hp transfection reagents in human breast cell lines. Ive learned a lot about how to improve my own lipofectamine 2000 mediated transfection.
Invitrogen lipofectamine 2000 transfection reagent life. I am trying to optimize the transfection by using lipofectamine 2000 for 2000 ng of plasmid dna well with different. Viafect reagent offers performance similar to or better than that of lipofectamine 2000 reagent, and it may be suitable for. Invitrogen lipofectamine transfection reagent protocols have been. Lipofectamine 2000 from invitrogen biocompare product. Invitrogen lipofectamine 2000 transfection reagent fisher. Exceptional transfection efficiency in the broadest range of cell lines and the highest levels of recombinant protein expression. 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. To transfect cells with sirna, follow the protocol as described for dna but. Cellspecific transfection protocols thermo fisher scientific fr. Luciferase expression was compared at 24 hours posttransfection using a standard luciferase assay. Original article an improved method for increasing the efficiency of.
Transfection commonly refers to the introduction of nucleic acids into eukaryotic cells, or more specifically, into animal cells. Lipofectamine 2000 transfection for hek293 cells about 6070% transfection rate transfect cells 6well plate. Lipofectamine 3000 80% transfection efficiency and 3 fold protein expression improvement over lipofectamine 2000 rnai transfection sirnamirna and gene silencing. Lipofectamine 2000 cd reagent is a proprietary animal originfree formulation for transfecting nucleic acids into eukaryotic cells. Dnaintm transfection reagent family a comparative study of transfection reagents. I want to try to use hela cell and lipofectamine 2000 to. Provided below are the esirna transfection conditions for some commonly used cell lines. Ive also used silentfect for sirna in u2os with 90% efficiency. Transfection efficiencies lower than 50% were not reported in this table. Transfection p19 cells with lipofectamine 2000 the same protocol can be used for 293t cells 1. Lipofectamine 2000 cd reagent complexes can be added directly to cells in culture medium. Lipofectamine 3000 reagent thermo fisher scientific kr.
Transfection protocol lipofectamine 3000 transfection reagent. The intracellular trafficking mechanism of lipofectamine based transfection reagents and its implication for gene delivery. Lipofectamine 2000 lf2000 is a reagent that i have found fits exactly what i need. A reverse transfection protocol was used to transfect mcf7 and hepg2. Add 100l of optimum to the eppendorf tube to dilute the dna and mix by tapping. Effi ciency and ofp expression were analyzed 72 hours posttransfection and a u2os and b hepg2 cells showed 4fold and 80fold improvement with lipofectamine. I want to try to use hela cell and lipofectamine 2000 to do. Invitrogen has taken the time to optimize this reagent and the recommend protocol that they provide. For additional information on scaling your transfection reaction, see page 4. I use 24well plates for transfections in triplicates. Table 2 examines transfection efficiency and cell viability under optimal conditions for each reagent 24 hours after transfection.
The highest transfection efficiency in many cell types and formats e. Optimizing transfection lipofectamine 2000 cd reagent. Cells were seeded in 96well flat bottom nontissue culture plate just before transfection with the density 50,000 cells per well in 100. Add 6l of lipofectamine2000 from biostores and 100 l of. 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. Dec 01, 2009 i use lipofectamine 2000 for u2os and usually get around 5070% efficiency in u2os for plasmids and 8090% efficiency of sirna. To transfect cells in different tissue culture formats, vary. 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. Can anyone suggest how much lipofectamine is optimal and after how much time i should add complete media. The trusted, simple, broadspectrum transfection reagent for most cell lines. Transfection the delivery of dna or rna into eukaryotic cells is a powerful tool used to study and control gene expression. Highest transfection efficiency in many cell types and formats. Transitx2 dynamic delivery system and lipofectamine 2000 transfection reagent were used to transfect plasmid dna encoding luciferase into 41 different cell types at three reagenttodna ratios.
Viafect reagent offers performance similar to or better than that of lipofectamine 2000 reagent, and it may be suitable for some cell lines where other lipidbased reagents result in lower transfection efficiencies table 1. Transfection protocol lipofectamine 3000 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. I am trying to do transfection in mammalian cells u2os.
Why do we have dead u2os cells on the perimeter of the wells in 6well plates upon lipofectamine rnaimax transfection. Using lipofectaminez 2000 for transfection provides the following advantages. Transfection guide overview of transfection methods promega. Dna ratio, but with the u2os i cant seem to be able to establish a. Theres a difference between the two protocols lipofectamine 3000 has an extra reagent that needs to be added. Maintain p19 cells undifferentiated in mem with 10% serum 7. 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. Cells for transfection were harvested at approximately 1,000,000 cells ml. Transfection guide overview of transfection methods. 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.
The goal of transfection optimization is to achieve maximal transfection efficiency with minimal cytotoxicity. Lipofectamine 2000 reagent thermo fisher scientific. I use lipofectamine 2000 for u2os and usually get around 5070% efficiency in u2os for plasmids and 8090% efficiency of sirna. However, for cotransfection of sirnachimera and plasmid dna, lipofectamine 2000 should be used. U2os, human osteosarcoma, sirna, lipofectamine rnaimax, 778075 778. Hek293 has since become a popular choice for recombinant protein expression and viral production due to its relatively easy maintenance as well as its amenability. Improved delivery of cas9 proteingrna complexes using. A b 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 invitrogen lipofectamine 3000 transfection reagent. Lipofectaminez 2000 is a proprietary formulation suitable for the transfection of nucleic acids into eukaryotic cells. Well, i obtain good results using lipofectamine2000, i dont know if they work in the. In a majority of cases, one wants the highest level of transfection possible with the lowest amount of cell death. We have developed a simple protocol to transfect mammalian cells using linear polyethylenimine pei.
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