Hek293 is a cell line derived from human embryonic kidney cells grown in tissue culture. Invitrogen lipofectamine 2000 transfection reagent fisher. The sameday transfection and plating or reverse transfection will also help boost your percent transfected. I use 24well plates for transfections in triplicates. Invitrogen lipofectamine 2000 transfection reagent 15ml. The lenti293t is an hek 293 derived cell line stably expresses the sv40 large t antigen and neomycin resistance gene. 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. Optimized conditions for successful transfection of human. Lipofectamine 2000 is commonly used for sirna transfections. Transient transfection of 293t cells faculty websites. 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.
As a result, the transfection procedure is identical to the transfection of freestyle 293f cells with the exception that cells are propagated and maintained in the 50. Though fairly efficient, we have found that being cationic, a negative charge can build up in the membrane as a result, making patchclamp. Improved delivery of cas9 proteingrna complexes using. 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. Scaling up or down lipofectamine 3000 reagent transfections use the following table to scale the volumes for your transfection experiment.
Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations. Lipofectamine 2000 cd reagent is a proprietary animal originfree formulation for transfecting nucleic acids into eukaryotic cells. Make sufficient 10% fbs in optimem media wo antibiotics sterile filter 2. 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. Other thing is to incease the cell density to 8595% confluncy as suggested by the manufacturer. Lipofectamine 2000 reagent thermo fisher scientific. It increases the likelihood of successful cleavage and recombination with talens or crisprs through highly efficient transfection, ultimately maximizing the efficiency of genetic modifications and simplifying the downstream processes. However, i could only reach at most 2030% transfection efficiency. Lipofectamine 2000 transfection reagent from invitrogen.
Advanced transfection with lipofectamine 2000 reagent. I agree with parvoman that one of the main considerations is how well the cells are growing. Researchers use lipofectamine 2000 reagent for sirna and shrnabased gene knockdown experiments, as well as for gene expression studies. Use a range of several ul of lipofectamine and ug dna to increase viability after transfection and efficiency. An improved method for increasing the efficiency of gene. Refer to the cell lines database for a list of cell types successfully. Transfection of 293 cells in 10 cm dishes abbreviated protocol surface area of 10 cm dish is 8 x greater than that of a 6well dish. This particular line was initiated by the transformation and culturing of normal hek cells with sheared adenovirus 5 dna. Exosomeliposome hybrid nanoparticles deliver crisprcas9. May 11, 2016 lipofectamine reagents are widely accepted as goldstandard for the safe delivery of exogenous dna or rna into cells.
To identify the best lipid nanoparticles for delivery of purified cas9 protein and grna complexes cas9 rnps into mammalian cells and to establish the optimal conditions for transfection. Lipofectamine 2000 represents one of the more recent cationic lipid encapsulation reagents which deliver dna to the cell by fusion and incorporation of lipid microsomes into the cells lipid bilayer. 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 amount of lipofectamine 2000 should be changed in proportion to the culture medium. After a 5 minute incubation in optimem, the lipofectamine 2000 and dna are combined and incubated for 20 minutes with gentle shaking at room temperature.
Using a systematic approach, we screened 60 transfection reagents using six commonlyused mammalian cell lines and identified a novel transfection reagent named lipofectamine crisprmax. Help on transfecting mcf7 and hct 116 cells oct222008 hi all. Mar 03, 2014 to determine the required amount of lipofectamine 2000 for forming of lipoplexes, different amounts of lipofectamine 2000, 1, 2, 4, 6. Each tube gets 8mls of optimem, 32ug of gag pol, 12. Canonical pathway enrichment analysis shows the primary biological processes impacted in hela cells that were transfected with transit. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. C the protein profile of hek293ft cells and the exosomes secreted by hek293ft cells were analyzed by silver blotting.
Hek293ft cellderived exosomes were isolated from the culture supernatants via polyethylene glycol peg 6000. Thee two most important factors in choosing transfecting reagent are. Lipofectamine 2000 procedure for simultaneously transfecting. Improving transfection efficiency of ultrapure oligochitosandna. Hek293 cell line origins, cytogenetics, and expression. Lipofectamine transfection of c2c12 cells bridges lab. Hek293ft cells were transfected with gfp vector pegfpn3 by polyjet left panel and lipofectamine 2000 right panel respectively. Label each tube with a different viral factor oct4, sox2, klf4, or cmyc. Hek293 cells, bj fibroblasts, and ecs were transfected with 1. Both lipofectamine 2000 and dna are mixed with optimem media and the quantity of each is dependant on the type of culture vessel i. Lipofectamine 2000 dna transfection reagent protocol. Hi, i tried to transfect my hek293t cells with pegfp using lipofectamine 3000, following manufacturers instructions.
Ive been using hek293ft cells, and lipofectamine 2000 for transfection with 2nd generation packaging system 2ug. Product is guaranteed for 6 months from the date of shipment if. Sep, 2005 both lipofectamine 2000 and dna are mixed with optimem media and the quantity of each is dependant on the type of culture vessel i. Mix optimem and lipofectamine in another eppendorf 32 l lipofectamine in 0,8 ml optimem 3. Add 6l of lipofectamine2000 from biostores and 100 l of. Theres a difference between the two protocols lipofectamine 3000 has an extra reagent that needs to be added. Although of epithelial origin, its biochemical machinery is capable of carrying out most of the posttranslational folding and processing required to generate functional, mature protein from a wide spectrum of both mammalian and nonmammalian nucleic acids. Strong expression of egfp was observed in hek293 cells, mouse. Effects of lipofectamine 2000sirna complexes on autophagy. Lipofectamine 2000 cd reagent complexes can be added directly to cells in culture medium. How to transfect hek293 cells using lipofectamine 3000. Depending on the size of culture vessel being used, the lipofectamine 2000 is diluted between 25 and 50fold.
Effi ciency and ofp expression were analyzed 72 hours posttransfection and a u2os and b hepg2 cells showed 4fold and 80fold improvement with lipofectamine. Lipofectamine 2000 reagent protocol 202 lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. Anyone else have toxicity issues with lipofectamine 2000. We use fugene6 with hek293 cells pretty routinely and get good results.
I would like to transfect 293t cells with lipofectamine 3000. 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. Lipofectamine 3000 reagent is suitable for novel genome editing applications. The hek cell line has been extensively used as an expression tool for recombinant proteins since it was generated over 25 years ago. Mix optimem with dna in an eppendorf 510 g dna0,8 ml optimem 2. Optimemin ul, equal volume for dna and lipofectamine lipofectaminein ul 25. The transformation resulted in the incorporation of approximately 4. Lipofectamine 2000 reagent is a lipidderived reagent designed to deliver exogenic nucleic acids dna andor rna into eukaryotic cells, either adherent or in suspension. 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 purpose of this study is to evaluate the effect of sirna transfection using lipofectamine 2000 on cellular autophagy. Lipofectamine 2000 transfection protocol 10cm plates reagents. Product is guaranteed for 6 months from the date of shipment if stored properly. Highest transfection efficiency in many cell types and formats e. Regarding cytotoxicity of hct116 cells, they at confluency 70% or higher can tolerate of 1.
Despite this, a satisfactory mechanismbased explanation of their. Effects of lipofectamine 2000sirna complexes on autophagy in. To determine the required amount of lipofectamine 2000 for forming of lipoplexes, different amounts of lipofectamine 2000, 1, 2, 4, 6. The reagent provides high transfection efficiency in many cells types and formats. Effects of lipofectamine 2000 sirna complexes on autophagy in hepatoma cells.
Cells in a 6 well dish, plated and at 70% confluence. Lipofectamine transfection of c2c12 cells bridges lab protocols. Cas9 protein and hprt1 grna were transfected into hek293 and hct116 cell lines. Date 20 july 2012 description lipofectamine 2000 transfection reagent is a proprietary formulation for transfecting nucleic acids dna, rna, and. High efficiency transfection reagent at a reasonable price. If you let them reach confluency, even if this happened a few passages ago, the transfection efficiency will be significantly diminished. Invitrogen lipofectamine 2000 transfection reagent.
The intracellular trafficking mechanism of lipofectamine. However, few studies have examined cellular responses to this delivery system. The transfection efficacy of lipofectamine 2000 was compromised by. Lipofectamine 2000 is a transfection reagent with liposome structure. Invitrogen lipofectamine 3000 transfection reagent. Lipofectamine contains lipid subunits that can form liposomes in an aqueous environment, which entrap the. They are also known, more informally, as hek cells. Lipofectamine 2000 reagent protocol 202lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. A comparison showing transfection efficiency of polyjet reagent vs. Representative image of three different experiments. Lipofectamine reagents are widely accepted as goldstandard for the safe delivery of exogenous dna or rna into cells. Hek293 cells were transiently transfected with lentivirus expression vector plenti cmvgfpdest separately using lipofectamine 3000, 2000 or. Flick each 50 ml conical tube and let sit for 5 minutes at room temperature.
For hek293, lipofectamine 3000, fugene and rnaimax showed the. Lipofectamine 2000 transfection reagent is versatile transfection reagent that has been shown to effectively transfect widest variety of adherent and suspension cell lines. As a result, the transfection procedure is identical to the transfection of freestyle 293f cells with the exception that. Lipofectamine 2000 and lipofectamine rnaimax transfection. The negative log of the p values is the probability of. Lipofectamine or lipofectamine 2000 is a common transfection reagent, produced and sold by invitrogen, used in molecular and cellular biology. Add 100l of optimum to the eppendorf tube to dilute the dna and mix by tapping.
Prepare the transfection buffer as noted in the material section. Pdf optimization of kyse30 esophagus cancer cell line. Protocol 6 well dish plate cells and grow to 70% confluence in 2 ml media without antibiotics dmem with 10% fbs, no psg. Combine diluted sirnachimera and lipofectamine 2000, vortex for a few second, incubated at room temperature for 20 min, then added to a 2ml culture.
Ask for a sample a comparison showing transfection efficiency of polyjet reagent vs. This is the first time i will have used lipofectamine 3000, previously i only used lipofectamine 2000, and i am wondering if anyone. Lipofectamine 2000 and lipofectamine rnaimax transfection reagents introduction life technologies is committed to providing researchers and scientists the best products, enabling cutting edge technology while keeping the environment in mind. Help on transfecting mcf7 and hct 116 cells transfection. Lipofectamine dna transfection reagents protocol see page 2 to view a typical plasmid transfection using a lipofectamine reagent. In some cases, that accompanies with reduced transfection efficiency, sometimes it does not. Original article an improved method for increasing the efficiency of. Transfection of nih3t3 cells, hela, swis 3t3, 293t with. Prewarm 50ml of optimum stored in cold room at 4c 10min.
Lipofectamine 2000 transfection for hek293 cells about 6070% transfection rate transfect cells 6well plate. Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. Prior to mixing with dna, lipofectamine 2000 should be diluted in optimem i. Pool dishes of cells that have been prepared for transfection. Lipofectamine 2000 reagent delivers dna or sirna with excellent transfection performance for protein expression, gene silencing, and functional assays. Scaling up or down lipofectamine 3000 reagent transfections. D the protein levels of alix, tsg101, cd63, cd81, cd9, and gapdh in hek293ft cell lysates and the exosomes secreted by hek293ft cells were analyzed by western blot. Lipofectamine 2000 transfection reagent dartmouth college. However, significant toxicity from lipofectamine 2000 was also reported in u2os cells.
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