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. Highest transfection efficiency in many cell types and formats e. Three cell densities of hepg2 cells were transfected with gapd sirna 100 nm using a range of volumes 0. Antibody quantities 7 days post transfection expressed by hek293 using either xellvivo tm or a reference medium. Do not add antibiotics to media during transfection as this causes cell death.
Altogen cro offers in vivo rnai services, tumor xenograft models, toxicology testing, stable cell line generation, and. However, i could only reach at most 2030% transfection efficiency. Hek293 cell line origins, cytogenetics, and expression. 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. This lipid reagent has proven to be effective for many different cultured mammalian cell lines. In addition, high transfection efficiency of hek293 cells produces exogenous proteins or viruses for pharmaceutical and biomedical research purposes. Transfection conditions for expi293f cells for each 30ml transfection, use 7. Invitrogen lipofectamine ltx reagent is a proprietary, animalorigin free formulation for the transfection of dna into eukaryotic cells with low cytotoxicity. Protocol 6 well dish plate cells and grow to 70% confluence in 2 ml media without antibiotics dmem with 10% fbs, no psg. For additional information on scaling your transfection reaction, see page 4. Comparison of fluorinated dendrimers g5f7 49 and g5f7 68 with g5 and lipofectamine 2000 on the in vitro egfp a,b and luciferase c gene transfection efficacy in hek293. To perform transfection experiments in other cell culture plates, simply multiply the suggested quantities by the relative surface area of your plate. Original article an improved method for increasing the efficiency of.
Prior to transfection, hek 293 cells are preincubated with serumfree dmem medium for 1 h. Hek2936e cell preparation for transfection 20101121jv. Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations. Plate an amount that will result in approximately 85 to 90% confluence see table below on the day of transfection. Rnai in cultured mammalian cells using synthetic sirnas. Transfection amounts component 96well 24well 6well dna per well 100 ng 500 ng 2500 ng plus reagent per well 0. Higher densities can lead to spontaneous differentiation. Our results suggest that lipofectamine 3000 outperforms lipofectamine 2000 and fugene hd in transfection efficiency andor protein expression, which is consistent with the claims of the manufacturer who reported it for hek293, hela, lncap, hepg2, and a549 cell lines, the superiority of lipofectamine 3000. We did not find substantial transfection efficiency variations in. 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. Two component lipidbased proprietary formulation optimized for hek293 cells transfection. Hek 293 cells are efficiently transfected with lipofectamine, with typically 70% of cells showing fluorescence following transfection with green fluorescent protein gfp. Hi, i tried to transfect my hek293t cells with pegfp using lipofectamine 3000, following manufacturers instructions.
Optimemin ul, equal volume for dna and lipofectamine lipofectaminein ul 25. Among them, although rnaimax displayed the second highest transfection efficacy, and it had the best safety profile, with 83. Aug 07, 2012 the day prior to your transfection, seed your cells so that they will be 70% to 90% confluent at the time of your experiment. Lipofectamine 2000 reagent protocol 202 lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. Lipofectamine 2000 dna transfection reagent protocol. Expi293f expression system kit thermo fisher scientific. Researchers use lipofectamine 2000 reagent for sirna and shrnabased gene knockdown experiments, as well as for gene expression studies. 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. Achieve superior transfection efficiency while maintaining low toxicity lipofectamine 3000 reagent maintains a high transfection efficiency within a robust dynamic range of lipid doses for quick and easy optimization. To obtain the highest transfection efficiency and low nonspecific effects, optimize transfection conditions by varying rna and lipofectamine 2000 concentrations. Thee two most important factors in choosing transfecting reagent are. Add 100l of optimum to the eppendorf tube to dilute the dna and mix by tapping.
Expifectamine 293 transfection kit protocol see page 7 to view a typical transfection procedure. Lipofectamine ltx dna transfection reagent protocol see page 2 to view a typical plasmid transfection procedure. For additional information, and protocols on transfecting suspension cells refer to the manual at. Hek293 transfection reagent epithelial kidney cells. For hek293, lipofectamine 3000, fugene and rnaimax showed the. Genscript recommends using lipofectamine 2000 for all transfections. Lipofectamine transfection of c2c12 cells bridges lab protocols. Lipofectamine 2000 transfection reagent from invitrogen. The optimal amount of dna to use in the transfection will vary widely depending upon the type of dna, transfection reagentmethod, target cell line, and number of cells genetic material dna quality and quantity factors affecting transfection 8. Seed cells onto the wellplates petridishes to be transfected so that day 1 is at. Invitrogens lipofectamine 2000 is one of many cationic lipidbased transfection reagents presently on the market. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. In hacat cells, lipofectamine 2000 achieved a transfection efficiency of 22. For 96 well plate for me worked well plating approximately cells well transfecting them with 300ng dna and 0.
In tube a place 2 g of dna in optimemi for a final volume of 200 l. It is recommended that cells are growing at 9095% confluence prior to transfection for adherent cells. Prepare plasmid dnalipid complexes recommend 2 doses of lipid. Several cell densities and lipid volumes were investigated to determine optimal transfection conditions, shown by the shaded boxes. Date 20 july 2012 description lipofectamine 2000 transfection reagent is a proprietary formulation for transfecting nucleic acids dna, rna, and. Laboratory 2010 lipofectamine 2000 transfection protocol 10cm plates reagents. The purpose of the guidelines below is to provide a procedure to generate hek293 6e cells that allow optimal transient protein expression. Hek293 6e cell preparation for transfection 20101121jv. I have ordered lipofectamine 2000 from thermo fishers scientific. Lipofectamine 2000mrna complex mix were immediately divided into 96well plates, at 104 cellswell for sknas and 5x103 cellswell for hek293 and incubated at 37. 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. This is necessary because serum is replete with rnases that degrade. Lipofectamine 2000 transfection reagent dartmouth college.
Invitrogen lipofectamine 3000 transfection reagent. Systematic screening of commonly used commercial transfection. Optimized conditions for successful transfection of human. Lipofectamine 2000 is used to deliver the sirnas into hek293 cells. L3000001 lipofectamine 3000 transfection reagent sparshbio. The transfection efficacy of lipofectamine 2000 was compromised by. Transfection reagents are highly efficient for dna and sirna transfection in vivo and in vitro. Lipofectamine 2000 reagent is a lipidderived reagent designed to deliver exogenic nucleic acids dna andor rna into eukaryotic cells, either adherent or in suspension. Method for efficient transfection of in vitrotranscribed. Cd 293, 293 sfm ii, and vpsfm are media formulations known to inhibit. In tube b place 190 l of optimemi and 10 l lipofectamine 2000. However, for cotransfection of sirnachimera and plasmid dna, lipofectamine 2000 should be used.
Transfection p19 cells with lipofectamine 2000 the same protocol can be used for 293t cells 1. Hek 293 cells are popular for their ease of growth and transfection hek293 transfection kit, making them a common cell culture in cancer research. 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. Before seeding cells for virus package, make sure hek293t cells are in rapid replication state by growth cells to approximately 75%80% confluence in a 10 cm cell culture dish and passaging at 1. Hek293 transfection reagent is supplied in liquid form at a concentration of 0. In biontex experiments gfp positivity increased to 90. With focus on high cell growth, transfection efficiency and productivity xellvivo tm led to a 4 fold increase in comparison to a commonly used reference medium. Transfection protocol of lipofectamine rnaimax can be obtained from the invitrogen website. Add 6l of lipofectamine2000 from biostores and 100 l of. Invitrogen lipofectamine 2000 transfection reagent.
Cells need to double with a 24 hr doubling rate to be ready for transfection d. Transfection efficiencies were optimal around 40% of transfected cells per spot with 11. This reference provides a recommended procedure to transfect plasmid dna into hek 293, human embryonic kidney cells atcc no. Lipofectamine 3000 transfection reagent leverages our most advanced lipid nanoparticle technology to provide superior transfection performance with improved application outcomes and reproducible results. Optimemin ul, equal volume for dna and lipofectamine lipofectamine in ul 25. This reagent delivers superior transfection efficiency and improved cell viability for the widest range of hardtotransfect and common cells e. Sep, 2005 lipofectamine 2000 is an easy to use transfection reagent. Altogen biosystems provides in vivo transfection reagents, over 100 preoptimized in vitro transfection kits for cell lines and primary cells, and electroporation delivery products. Lipofectamine 2000 transfection reagent is versatile transfection reagent that has been shown to effectively transfect widest variety of adherent and suspension cell lines. Ideally have the cells double two consecutive times at maximum doubling rate log phase before transfection e.
Lipofectamine 2000 transfection protocol 10cm plates reagents. At 24 h after transfection, the transfection efficiency was evaluated using fluorescence microscopy, by estimating the. Help on transfecting mcf7 and hct 116 cells transfection. Lipofectamine 2000 is a transfection reagent with liposome structure. Can anyone provide me with a proper transfection protocol into hek293 cells with lipofectamine 2000. Lipofectamine is formulated to give high transfection efficiency with little toxicity to cells. Pdf optimization of kyse30 esophagus cancer cell line. Lipofectamine 2000 for transfection provides the following advantages. Hek293 transfection reagent is tested functionally by transfection of hek293 cells with a small. Transient plasmid dna transfection into hela and hacat.
Invitrogen lipofectamine 2000 transfection reagent fisher. Genetic transfection an overview sciencedirect topics. To transfect cells with sirna, follow the protocol as. Transfection of nih3t3 cells, hela, swis 3t3, 293t with. If using reagents suitable for the transfection of dna and rna, transfection of a plasmid that encodes gfp is a convenient way to test and calibrate efficiencies. A guide to transient expression of membrane proteins in. The media needs to be changed at least 1hr prior to transfection. Maintain p19 cells undifferentiated in mem with 10% serum 7.
Hek293 cells, bj fibroblasts, and ecs were transfected with 1. Plate cells so they will be 7090% confluent at the time of transfection. Can anyone provide me with a proper transfection protocol. 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. Theres a difference between the two protocols lipofectamine 3000 has an extra reagent that needs to be added. The day prior to your transfection, seed your cells so that they will be 70% to 90% confluent at the time of your experiment. From the luciferase activities and consistencies of data using different transfection reagents including genejammer, effectine, wellfect, lipofectamine, what i can say from the data i observed, the efficiency of transfection is much more better in hct116 cells. We did not find substantial transfection efficiency variations in different batches of lipofectamine 2000.
Make sufficient 10% fbs in optimem media wo antibiotics sterile filter 2. Lipofectamine 2000 reagent thermo fisher scientific. Hek293 6e cell preparation for transfection 20101121jv purpose. Lipofectamine 3000, rnaimax, fugene and lipofectamine 2000 all displayed high transfection efficacy 97. 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. Lipofectamine transfection of c2c12 cells bridges lab. Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. Scale your transfections up or down by proportionately adjusting the amounts of the reagents used. I use 24well plates for transfections in triplicates. Invitrogen claims that this reagent gives the highest protein expression levels in the widest variety of adherent and suspension.
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