This reference provides a recommended procedure to transfect plasmid dna into hek 293, human embryonic kidney cells atcc no. Add 100l of optimum to the eppendorf tube to dilute the dna and mix by tapping. Expi293 expression system thermo fisher scientific. Lipofectamine 2000, and lipofectamine 2000 cd x broad spectrum dna deliveryachieve high expression in many cell types, including hardtotransfect cells and insect cells x animal origin freetransit2020 provides high performance with maximum compatibility description transit2020 reagent is a new and versatile transfection. To transfect cells in different tissue culture formats, vary the amounts of lipofectamine 2000, nucleic acid, cells, and medium used in proportion to the relative surface area, as shown in the table. Lipofectamine ltx reagent is a proprietary, animalorigin free formulation for the transfection of dna into eukaryotic cells with low cytotoxicity. Theres a difference between the two protocols lipofectamine 3000 has an extra reagent that needs to be added.
For each transfection sample, prepare dna lipofectamine 2000. Lipofectamine 2000 transfection reagent is versatile transfection reagent that has been shown to effectively transfect widest variety of adherent and suspension cell lines. Lipofectamine 2000 is that it has an extensive, very useful manual. Transfection conditions for expi293f cells for each 30ml transfection, use 7. After incubation, remove media and replace with fresh media. 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. Add prepared transfection complexes from step 3 or 4 7. Lipofectamine 2000 transfection for hek293 cells about 6070% transfection rate transfect cells 6well plate. Transfection protocol lipofectamine 3000 transfection reagent.
Can anyone provide me with a proper transfection protocol. Hek 293 cells are efficiently transfected with lipofectamine, with typically 70% of cells showing fluorescence following transfection with green fluorescent protein gfp. Use this brief procedure to transfect stealth rnai or sirna into mammalian cells. Refer to the cell lines database for a list of cell. Gibco hams f12 nutrient mix 11765054 5% horse serum 1 mm gibco sodium pyruvate 160070 25 mm gibco hepes 15630106 proper culture techniques and procedures are an essential. Hi, i tried to transfect my hek293t cells with pegfp using lipofectamine 3000, following manufacturers instructions. Perform reverse transfection by preparing complexes inside the wells, and then adding cells. After 5 min, diluted dna was added to diluted lipofectamine 2000 and incubated for 15 min. Cells should reach 5080% confluency at time of 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. Lipofectamine transfection of c2c12 cells bridges lab. Each expi293 expression system kit provides cells, culture medium, and reagents to express 1 liter of cell. Optimemin ul, equal volume for dna and lipofectamine lipofectamine in ul. The trusted, simple, broadspectrum transfection reagent for most cell lines.
Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. Aspirate the medium from the dish of cells, add the diluted transformation mix, and incubate the cells with the mix in a 10% co 2 incubator for approximately 5 h. Original article an improved method for increasing the efficiency of. Maintain p19 cells undifferentiated in mem with 10% serum 7.
I transiently transfect hek cells with plasmids which encode an ion channel. The transfection efficiency with lipofectamine plus was 82% with a viability of. Transfection reagents are highly efficient for dna and sirna transfection in vivo and in vitro. The main advantage of using lipofectamine 2000 however, was its ability to transfect a range of very different cell types coupled with a high efficiency of transfection. I am currently having problems with my transfections and request help. 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. Combine diluted sirnachimera and lipofectamine 2000, vortex for a few second, incubated at room temperature for 20 min, then added to a 2ml culture. Arpe19 transfection kit retinal pigment epithelium.
Hi, i have a question about the use of lipofectamine 2000 for transfection of 293t cells. Lipofectamine 2000 is used to deliver the sirnas into hek293 cells. This allows the dna and the lipofectamine to form the complexes that will get the dna into the cells. My lab is looking for an alternate transfection reagent to fugene 6 for transient transfection with minimal toxicity into hek 293 cells. For each transfection sample, prepare dnalipofectamine 2000 complexes as follows. Lipofectamine 2000 dna transfection reagent protocol see page 2 to view a typical dna transfection procedure. Transfection protocol lipofectamine 3000 transfection reagent lncap prostate cancer cells complete growth medium component cat.
Lipofectamine 2000 transfection reagent dartmouth college. Lipofection transfection of hek293 cells unsuccessful. So i did the tranfection, followed up by western blotting. Transfection of human hek 293f cells with metafectene pro. Using cx43 shrna lentivirus combined with lipofectamine 3000 transfection. The transfection efficiency of neutral and plasmidloaded cationic mbs was then compared with that of lipofectamine 2000 for various adherent cell lines. Lipofectamine 2000 reagent thermo fisher scientific.
I use 24well plates for transfections in triplicates. I found many cells detach easily after 6h incubation with lipofectamine 2000. Oct 20, 2009 i start working on hek293 cells recently. The gibco expi293 expression system is a highyield transient expression system based on suspensionadapted human embryonic kidney hek cells. Similar results were obtained on transfection of hek 293f cells with lipofectamine 2000 invitrogen. For additional information on scaling your transfection reaction, see page 4. The transfection efficacy of lipofectamine 2000 was compromised by. Lipofectamine 2000 cd reagent complexes can be added directly to cells. Transfection of nih3t3 cells, hela, swis 3t3, 293t with. If the transfected dna contains a fluorescent protein, check for transfection efficiency by viewing in a fluorescent microscope. The transfection efficiency and viability with lipofectamine 2000 was 98% and 67%, respectively.
L of cell culture medium without antibiotics so that they will be 30%50% confluent at the time of transfection. Lipofectamine 2000 cd reagent is a proprietary animal originfree formulation for transfecting nucleic acids into eukaryotic cells. Transfection efficiencies lower than 50% were not reported in this table. Comparative transfection of dna into primary and transformed. Transfection guide overview of transfection methods promega. Lipofectamine 2000 cd reagent complexes can be added directly to cells in culture medium. Plate cells for transfection only 1 day before the experiment. A simple and efficient method for transfecting mouse.
Altogen biosystems provides in vivo transfection reagents, over 100 preoptimized in vitro transfection kits for cell lines and primary cells, and electroporation delivery products. Transfection and genome engineering fisher scientific. Fugene 6 works great, but is very expensive and poses a. Scale your transfections up or down by proportionately adjusting the amounts of the reagents used. L reverse transfection of rnai reverse transfection is faster to perform than forward transfection and is the method of choice for highthroughput transfection. I use lipofectamine 3000 to transfect hek 293 cells in 6 wells plates. I want to do a transfection to overexpress protein bob in 293t cells. Using cx43 shrna lentivirus combined with lipofectamine 3000 transfection reagent, we can achieve about 90% cx43 knockdown efficacy in huvecs.
Rnai in cultured mammalian cells using synthetic sirnas. Serumfree medium is necessary for successful dnalipofectamine 2000. At 1224 h before transfection, plate the cells in 500. I am trying to optimize the transfection by using lipofectamine 2000 for 2000 ng of plasmid dna well with different amounts of lipofectamine 2000 5ul, 10ul in the 6well plate. Optimizing transfection lipofectamine 2000 cd reagent. My main purpose is to show that my antibody for bob works. Prior to transfection, hek 293 cells are preincubated with serumfree dmem medium for 1 h. Component 96well 24well 6well final dna per well 100 ng 500 ng 2500 ng. This is necessary because serum is replete with rnases that degrade. Invitrogen lipofectamine ltx reagent is a proprietary, animalorigin free formulation for the transfection of dna into eukaryotic cells with low cytotoxicity. Lipofectamine 2000 transfection reagent from invitrogen. Dilute appropriate amount of lipofectamine 2000 by optimem i reduced serum medium to 50 l and vortex for a few seconds for siha and skov3 cells, dilute 1. Gently mix media on cells via figure 8 pattern and back and forth motions. Gibco rpmi 1640 medium, with glutamax supplement 61870036 10% gibco fbs a3160401 1 mm gibco sodium pyruvate 160070 proper culture techniques and procedures are an essential part of ensuring successful.
Dna is enclosed within the liposomes and the complexes fuse with the host cell membrane for dna entry into the cytoplasm and eventu ally into the nucleus. 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. The transfection efficiency with lipofectamine plus was 82% with a viability of 80% data not shown. Method for efficient transfection of in vitrotranscribed. Cells in a 6 well dish, plated and at 70% confluence. The cell numbers required for the transfection depend strongly on the growth rate and the size of the cells. This lipid reagent has proven to be effective for many different cultured mammalian cell lines. Hi, i was wondering if you transfect the 293 cells in suspension or adherent as i normally gain about 7090% transfection using my protocol with cmvgfp and saintmix reagent. Lipofectamine 2000 transfection protocol 10cm plates reagents. May 11, 2016 lipofectamine reagents are widely accepted as goldstandard for the safe delivery of exogenous dna or rna into cells. Altogen cro offers in vivo rnai services, tumor xenograft models, toxicology testing, stable cell line generation, and.
Jul 19, 2016 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. Invitrogen lipofectamine 2000 transfection reagent. The intracellular trafficking mechanism of lipofectamine. Transfection commonly refers to the introduction of nucleic acids into eukaryotic cells, or more specifically, into animal cells. Good news, my antibody works pretty well tested 293t untransfected, transfected 293 two different clones, and psirpg transfected cells transfection. For adherent cells, we recommend 9095% confluence at the time of transfection for high efficiency, high expression levels, and to minimize cytotoxicity.
Cloned genes can be transfected into cells for biochemical characterization, mutational analyses, investigation of the effects of gene expression on cell. Despite this, a satisfactory mechanismbased explanation of their. How to transfect hek293 cells using lipofectamine 3000. Transient transfection of mammalian cells with lipofectamine. General procedure for transfection of mammalian cells with. Lipofectamine 2000 is tested for absence of microbial contamination with blood agar plates, sabaraud dextrose agar plates, and fluid thioglycolate medium, and functionally by transfection of chok1 cells with a reporter plasmid. By using newly trypsinized adherent cells in suspension and lipofectamine 2000, we detected a transfection efficiency of 8090% in both cell lines and a cell viability of 90% in sknas and 60% in hek293, 24 h after transfection. Lower confluence will achieve higher percentage of transfection but may yield a lower absolute number of transfected cells.
Prepare plasmid dnalipid complexes recommend 2 doses of lipid. I want to try to use hela cell and lipofectamine 2000 to. Expi293f expression system kit thermo fisher scientific. Prewarm 50ml of optimum stored in cold room at 4c 10min. Evaluation of transfection methods for transient gene. Incubate at 37c for 4 to 6 hours for delicate cells or overnight for tough cells. 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.
A hela cells were transfected with hcv dsrna using lipofectamine 2000 reagent. Lipofectamine 2000 is useful for transfecting dna plasmid. Lipofectamine 3000 that displayed strong toxicity to hek293 cells. After concentrating cells to such high densities, transfection of hek 293 cells becomes possible in most commonly used media and is not restricted to a specific medium. Transfection efficiencies were optimal around 40% of transfected cells per spot with 11. The reagent provides high transfection efficiency in many cells types and formats. Add the lipofectaminedna mixture to each well and gently swirl to mix.
However, i could only reach at most 2030% transfection efficiency. Increased transfection efficiency by the directed transport, especially for low amounts of nucleic acids high transfection rates for adherent mammalian cell lines and primary cell cultures suspension cells and cells from other organisms also successfully transfected but need to be immobilized mild treatment of cells. Count the cells using standard trypan blue exclusion. Hek293 cells detach after transfection scientist solutions. On the day of transfection just prior to preparing complexes, plate 1. Lipofectamine 2000, and lipofectamine 2000 cd x broad spectrum dna deliveryachieve high expression in many cell types, including hardtotransfect cells and insect cells x animal origin freetransit2020 provides high performance with maximum compatibility description transit2020 reagent is a new and versatile transfection solution for. Hek293 transfection reagent is supplied in liquid form at a concentration of 0. Make sufficient 10% fbs in optimem media wo antibiotics sterile filter 2. Researchers use lipofectamine 2000 reagent for sirna and shrnabased gene knockdown experiments, as well as for gene expression studies. Add 6l of lipofectamine2000 from biostores and 100 l of.
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. Transfection the delivery of dna or rna into eukaryotic cells is a powerful tool used to study and control gene expression. Original article an improved method for increasing the. Lipofectamine 2000 reagent delivers dna or sirna with excellent transfection performance for protein expression, gene silencing, and functional assays. Plate cells so they will be 7090% confluent at the time of transfection. 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. Invitrogen lipofectamine 2000 transfection reagent is a versatile transfection. Hek293 transfection reagent is tested functionally by transfection of hek293 cells. Lipofectamine 2000 dna transfection reagent protocol.
Higher densities can lead to spontaneous differentiation. For some cell types, such as cos7, the transfection efficiency was over 95% while other hardertotransfect cells. Lipofectamine 2000 from invitrogen biocompare product. Lipofectamine 2000 is a cationlipid formulation transfection reagent that forms liposomes in aqueous media. Transfection p19 cells with lipofectamine 2000 the same protocol can be used for 293t cells 1. A guide to transient expression of membrane proteins in hek. Two component lipidbased proprietary formulation optimized for hek293 cells transfection. The microscopic analysis was performed using antirigi mab. Seeding cells for transfection the day before transfection, dissociate cells that are 8090% confluent in a t75 flask.
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