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. Transfection the delivery of dna or rna into eukaryotic cells is a powerful tool used to study and control gene expression. Transfection of nih3t3 cells, hela, swis 3t3, 293t with. For gene silencing, lipofectamine 2000 transfection reagents highefficiency transfections provide the high levels of gene knockdown needed to produce. Lipofectamine 2000 is a very efficient transfection agent, the stability of your knockdown or your overexpression is only related to the half life of your sirna, plamid in the cell. If there appears to be largescale loss of cells after lipofectamine treatment, then it might pay to try using polylysinecoated plates. Following transfection the cells were cultured in 25 ml serum containing medium. Lipofectamine 3000 reagent was developed for high efficiency transfection and improved cell viability in the broadest range of cell types. Preliminary study of a novel transfection modality for in vivo sirna. Transfection was measured by luciferase activity using a conventional assay. Theres a difference between the two protocols lipofectamine 3000 has an extra reagent that needs to be added. To transfect cells with sirna, follow the protocol as described for dna but. 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. Cells should be plated on collagencoated dishes, as for normal propagation.
Each reagent was used to transfect hek 293, hela, lncap, a549, and hepg2 cells in 96well format. I use 24well plates for transfections in triplicates. Department of neurobiology and anatomy, university of utah school of medicine, salt lake city, utah. This is the first time i will have used lipofectamine 3000, previously i only used lipofectamine 2000, and i am wondering if anyone. To perform transfection experiments in other cell culture plates, simply multiply the suggested quantities by the relative surface area of your plate. From the above data in table 3, this indicates that the 1. May 11, 2016 lipofectamine reagents are widely accepted as goldstandard for the safe delivery of exogenous dna or rna into cells. Lipofectamine 2000 transfection reagent from invitrogen. Genscript recommends using lipofectamine 2000 for all transfections.
The weight ratio of lipofectamine 2000 to pdna was kept constant at 3. The amount of lipofectamine 2000 reagent required for successful transfection varies depending on the cell type and passage number. Lipofectamine is formulated to give high transfection efficiency with little toxicity to cells. Moreover, there is a high risk of degradation in complete serum if nucleic acids are not well protected.
I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. Freestyle 293f cells were transfected at a cell density of 1 x 106 cellsml in a 60 ml shaker flask using 293fectin and the freestyle protocol. Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. In fig 1 below there is very high level of low level transfection and there are also high levels of high. Lipofectamine reagent recognition publications citing lipofectamine 2000 reagent steadily rose after the market introduction in 1999, and this product continues to be one of the mostcited transfection reagents on. The product manual includes advice on how to optimize transfection efficiency, as a number of. Reverse transfection is the transfer of genetic material into cells and is reverse because the order of dna and cells is reverse that of conventional transfection. I would like to transfect 293t cells with lipofectamine 3000. Cells in a 6 well dish, plated and at 70% confluence. Lipofectamine, a popular in vitro transfection reagent composed of cationic lipid. Effects of lipofectamine 2000sirna complexes on autophagy in hepatoma cells. High gfp expression following transfection using lipofectamine 2000 reagent. For 96 well plate for me worked well plating approximately cells well transfecting them with 300ng dna and 0.
In some cases, that accompanies with reduced transfection efficiency, sometimes it does not. Highest transfection efficiency in many cell types and formats e. Advanced transfection with lipofectamine 2000 reagent. Despite this, a satisfactory mechanismbased explanation of their. Protocol 1 stable transfection using lipofectamine 2000 in 6 well plates. Higher densities can lead to spontaneous differentiation. Optimizing stealth rnai or sirna transfection to obtain the highest transfection efficiency and low nonspecific effects, optimize transfection conditions by varying rna and lipofectamine 2000 concentrations. Lipofectamine 2000 transfection reagent dartmouth college. Stealthsirna transfection protocol lipofectamine 2000. Add 6l of lipofectamine2000 from biostores and 100 l of. For all transfection of multiple wells in 6 well plate, multiply cdna, optimem, and lipofectamine quantity used by number of wells to transfect. We appreciate greatly the technical service of thermofisher and find them an. Transfection efficiencies using commercially available reagents such as fugene hd and lipofectamine 2000 were generally low, but even the best. Availability of an efficient transfection protocol is the first determinant in success of gene transferring studies in mammalian cells which is accomplished experimentally for every single cell type.
Invitrogen lipofectamine 3000 transfection reagent 0. Lipofectamine reagents are widely accepted as goldstandard for the safe delivery of exogenous dna or rna into cells. To obtain the highest transfection efficiency and low cytotoxicity, optimize transfection conditions by varying cell density as well as dna and lipofectamine 2000 concentrations. Lipofectamine 2000 reagent thermo fisher scientific. One day before transfection, plate cells in growth medium without antibiotics so that they will be 9095% confluent at the time of transfection 0. The intracellular trafficking mechanism of lipofectamine. Anyone else have toxicity issues with 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 mechanism of lipofectamine 2000 mediated transmembrane. Does lipofectamine 2000 give a stable transfection. It consistently produces high transfection efficiency and high protein overexpression. Add 100l of optimum to the eppendorf tube to dilute the dna and mix by tapping. Lipofectamine 2000 reagent is a lipidderived reagent designed to deliver exogenic nucleic acids dna andor rna into eukaryotic cells, either adherent or in suspension.
Make sufficient 10% fbs in optimem media wo antibiotics sterile filter 2. I found this possibility in lipofectamine 2000 manual, but not in lipofectamine 3000 manual. The transfection efficiencies of the pte cells were 5% effectene. All transit dna transfection reagents, including transitx2 dynamic delivery system, can be used for reverse transfection of dna into multiple cell types. Other thing is to incease the cell density to 8595% confluncy as suggested by the manufacturer. Antibodies secondary antibodies elisa kits assay kits biomolecules. Manual solid phase peptoid synthesis of liptoid was conducted according to.
Start any new transfection by testing the recommended four concentrations of lipofectamine 2000 reagent to determine an optimum amount. Lipofectamine 3000 reagent maintains a high transfection efficiency within a robust dynamic range of lipid doses for quick and easy optimization. Invitrogen lipofectamine 3000 transfection reagent from. Lipofectamine transfection of c2c12 cells bridges lab. Laboratory 2010 lipofectamine 2000 transfection protocol 10cm plates reagents. Thee two most important factors in choosing transfecting reagent are. While, the wet method has higher transfection efficiency as the premixing of dna with the transfection reagent lipofectamine 2000 facilitates the formation of the dnaliposome complex. Cells in a 6 well dish, plated and at 90% confluence. Lipofectamine 2000 reagent protocol 202lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. Lipofectamine 2000 dna transfection reagent protocol. Make sure that cells are greater than 90% confluent and vary dna. Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations. For adherent cells, we recommend 9095% confluence at the time of transfection for high efficiency, high. Lipofectamine reagent recognition publications citing lipofectamine 2000 reagent steadily rose after the market introduction in 1999, and this product continues to be one of the mostcited transfection reagents on the market figure 3.
Lipofectamine, lipofectamine 2000, and lipofectamine enhan. Transfection p19 cells with lipofectamine 2000 the same protocol can be used for 293t cells 1. 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. The transfection efficacy of lipofectamine 2000 was compromised by its high toxicity. Refer to the cell lines database for a list of cell types successfully. More importantly, on chip high throughput virus production combined with in situ target cell infection was achieved.
Maintain p19 cells undifferentiated in mem with 10% serum 7. Lipofectamine or lipofectamine 2000 is a common transfection reagent, produced and sold by invitrogen, used in molecular and cellular biology. How does transfection with lipofectamine 2000 work. 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. The lenti293t is an hek 293 derived cell line stably expresses the sv40 large t antigen and neomycin resistance gene. Max % of positive cells after transfection of a gfpplasmid 3. Fectopro transfection reagent for bioproduction polyplus. Comparison of three different transfection methods biology. Cut plasmid with an appropriate restriction enzyme to produce a linear fragment for transfection. Prewarm 50ml of optimum stored in cold room at 4c 10min. Lipofectamine plasmid transfection bridges lab protocols. 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. Lipofectamine contains lipid subunits that can form liposomes in an aqueous environment, which entrap the.
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. Sep, 2005 invitrogens lipofectamine 2000 is one of many cationic lipidbased transfection reagents presently on the market. However, for cotransfection of sirnachimera and plasmid dna, lipofectamine 2000 should be used. B hela cells were transfected with luciferase encoding plasmid dna using either transit. Effects of lipofectamine 2000sirna complexes on autophagy. 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.
Optimization of transfection methods for huh7 and vero. Lipofectamine 2000 transfection reagent thermo fisher scientific. Invitrogen lipofectamine 3000 transfection reagent. 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. Generally need to have 2 g of dna for each tranfection.
Branski rc,0000000311909036, nyu school of medicine. Enable high transfection efficiency in novel genome editing applications. Lipofectamine 2000 reagent protocol 202 lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. Lipofectamine 2000 transfection protocol 10cm plates reagents. Refer to the cell lines database for a list of cell types successfully transfected.
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. Lipofectamine 3000 showed the third highest transfection efficacy 37. Lipofectaminez 2000 for transfection provides the following advantages. Lipofectamine 3000 thermo fisher lipofectamine 3000 10. Invitrogens lipofectamine 2000 is one of many cationic lipidbased transfection reagents presently on the market. Mar 27, 2007 lipofectamine 2000 reagent is a lipidderived reagent designed to deliver exogenic nucleic acids dna andor rna into eukaryotic cells, either adherent or in suspension. Lipofectamine 3000 provided gentle but efficient transfection in several line of interest to our lab, heioc1 mouse cochlear 69fold, nccit and ntera2 3 fold testicular cancer lines and allowed for very nice dose response assays for cisplatin sensitvity assays. It is used to increase the transfection efficiency of rna including mrna and sirna or plasmid dna into in vitro cell cultures by lipofection. This protocol is for transfecting plasmid dna into mammalian cells. Transfection protocol of lipofectamine rnaimax can be obtained from the invitrogen website.
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