Since their initiation, hela cells have been used in transfection experiments all over the world to study viruses. I do have cell toxicity, but not so serious as yours. Hela transfection information and resources hela cell line. Lipofectamine 2000 right panel around 5% efficiency. The mechanism of lipofectamine 2000 mediated transmembrane. Protocol 6 well dish plate cells and grow to 70% confluence in 2 ml media without antibiotics dmem with 10% fbs, no psg. Lipofectamine tm 2000 invitrogen was used to aid the transfection of hela cells with the c 24. To perform transfection experiments in other cell culture plates, simply multiply the suggested quantities by the relative surface area of your plate. Using various chemical or physical methods, this gene transfer technology enables the study of gene function and protein expression in a cellular environment.
Transfecting primary cells in suspension protocol online. The intracellular trafficking mechanism of lipofectamine. Do you have a toxicity problem with lipofectamine 2000. Thermo scientific nunc 24well cell culture treated multidishes 142475 invitrogen lipofectamine 3000 transfection reagent l3000008 gibco optimem i reduced serum medium 31985062 step tube complexation components amount per well 24well. Twentyfour hours from the start of transfection, all wells were stained for. Protocol for dna transfection and luciferase assay using pfr. Youd want to place enough cells to make your transfection well 35%45% confluent since you know that they will double to 70%90% confluent in 24 hours. Conclusions in both hela and hacat cell lines, biontex k2 reagent afforded a higher transfection efficiency than lipofectamine 2000. Make sufficient 10% fbs in optimem media wo antibiotics sterile filter 2. Grow li cells transformed with pfrwt plasmid dna in appropriate volume of lb or 2xyt media with ampicillin overnight and isolate dna using standard qiagen mini or maxiprep protocol.
A sample protocol is listed here for transfection experiments performed in 6well plates. Lipofectamine ltx reagent is a proprietary, animalorigin free formulation for the transfection of dna into eukaryotic cells with low cytotoxicity. You can not use antibiotics during transfection step because it will cause cell death. Jul 17, 2007 lipofectamine 2000 is a lipid based transfection reagent for eukaryotic cells. Transient plasmid dna transfection into hela and hacat. But unfortunately the survival rate of cells has only been 46,9 61,9 %.
Prewarm 50ml of optimum stored in cold room at 4c 10min. Wash the cells 2x pbs seed 10 6 cells in 300 ul warm optimem in 24 wellsplates. In the protocol described here, we use a pool of three sirna duplexes per target. A protocol for efficiently transfecting mouse escs, our lab has been using a lf2k protocol adapted from bugeon et.
What they truly care about is working in harder to transfect cell lines, biologically. The total volume of the transfection mixes was 100ul, and it was added to the medium already in the wells. Mix 1 and 2 by adding the dna to lipofectamins dropwise. Optimized conditions for successful transfection of human. Lipofectamine 2000 transfection reagent is best transfection reagent available from life technologies for the cotransfection of sirna and plasmid dna. I want to try to use hela cell and lipofectamine 2000 to. Transfection and genome engineering fisher scientific. Lipofection is most widely used method for transfecting neurons for live cell imaging experiments. Since our lab routinely uses pei to efficiently transfect standard cell lines, such as hek293t, hela and neuro2a unpublished data, we wanted to see if pei could also be used to transfect mouse escs. Transient plasmid dna transfection into hela and hacat cells. Hela cells, for example, seem quite sensitive again, this depends on the batch. Biolistic particle delivery employs gold particles that conjugate with nucleic acids 16, 17.
Whats the difference between lipofectine and lipofectamine 2000 reagents. Theres a difference between the two protocols lipofectamine 3000 has an extra reagent that needs to be added. Genscript recommends using lipofectamine 2000 for all transfections. I use a gfp construct as a control for transfection. 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 2000 from invitrogen biocompare product. Invitrogen lipofectamine 3000 transfection reagent 1. All amounts and volumes are given on a per well basis. Original article an improved method for increasing the efficiency of. Used the same reagent and methods including wash steps and plasmid on u87 cells and had very bad results high toxicity with normally adherent cells in suspension. Choosing the right transfection reagent for optimal efficiency.
Lipofectamine 2000 reagent is a lipidderived reagent designed to deliver exogenic nucleic acids dna andor rna into eukaryotic cells, either adherent or in suspension. Plate cells so they will be 7090% confluent at the time of transfection. Transfect cells at high cell density for high efficiency, high. Transfection is the process of introducing nucleic acids into eukaryotic cells by nonviral methods. Transfection protocol lipofectamine 3000 transfection. Transfection is the process of introducing foreign dna into the cells either by physical. We did not find substantial transfection efficiency variations in different batches of lipofectamine 2000. Can anyone provide me with a proper transfection protocol. Dna complexes were mixed with cells in suspension and the mixture was then added to the empty wells fig.
The weight ratio of lipofectamine 2000 to pdna was kept constant at 3. Higher densities can lead to spontaneous differentiation. Nov 20, 2008 i have a question regarding confluency of hek293 cells for transfection. The amount of lipofectamine 2000 reagent required for successful transfection varies depending on the cell type and passage number. Start any new transfection by testing the recommended four concentrations of lipofectamine 2000 reagent to determine an optimum amount. Scaling up or down lipofectamine 3000 reagent transfections use the following table to scale the volumes for your transfection experiment.
It can be used for transfection of adherent or suspension cells with dna or rna including stealth rnai and sirna and can be used for either transient transfections or to create stable cell lines. Lipofectamine 2000 transfection reagent from invitrogen. 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. For instance, in electroporation procedure not only the level of cell. Or ideally youd place a known quantity of cells from a suspension culture since many fewer of them will be dead than after passage from a tflask. In lipofectamine 2000 transfection experiments 4 l of lipofectamine 2000 invitrogen was mixed with 100 l of optimem i gibco, incubated for 5 minutes at 25 c and then added to 100 l optimem i containing 2 g of plasmid dna. Lipofectamine 2000 transfection protocol 10cm plates reagents. Hela tranfection dual luciferase assay tissue and cell. What i found is lipofectamine 2000 is toxic to cells, so i reduced the recommended quantity of the invitrogen protocol.
I get consistent results using this lipid for my transfections. Does it mean that once hek293 cells reached 100% confluency, it cannot be used at all for transfection expts. It was the hela cell line that took up the polio virus and. Many transfection protocols require serumfree conditions for optimal. Does anyone have experience with the lipofectamine 2000. Transfection p19 cells with lipofectamine 2000 the same protocol can be used for 293t cells 1. Lipofectamine transfection of c2c12 cells bridges lab. Hela transfection is usually pretty standard in terms of protocols just make sure.
Lipofectamine or lipofectamine 2000 is a common transfection reagent, produced and sold by invitrogen, used in molecular and cellular biology. Wash cells with two volumes of dpbs and add fresh complete rpmi media. Total rna levels were decreased in samples transfected with lipofectamine 2000 for 24 hours. If the transfected dna contains a fluorescent protein, check for transfection efficiency by viewing in a fluorescent microscope. Maintain p19 cells undifferentiated in mem with 10% serum 7. I want to try to use hela cell and lipofectamine 2000 to do. Transfection efficiencies were optimal around 40% of transfected cells per spot with 11. To optimize the transfection into hela cells by lipofectamine tm 2000, hela cells were seeded 16 hours before transfection onto a 6well plate at four cell densities of 104, 2. Lipofectamine reagents are widely accepted as goldstandard for the safe delivery of exogenous dna or rna into cells. I have used lf2000 successfully with the following cell lines. A rna was harvested from hela cells that were transfected with a non.
Invitrogen lipofectamine 2000 transfection reagent strong performance in a wide range of cell lines reliableone of the mostcited dna reagents strong performancefor easytotransfect to moderately difficulttotransfect cell lines easy to usesimple protocol for easy optimization in a. Advanced transfection with lipofectamine 2000 reagent. Jun, 2010 this method delivers nucleic acids into cells but demands skill, often causes cell death, and is very laborintensive. Invitrogen lipofectamine 3000 transfection reagent l3000008 gibco optimem i reduced serum medium 31985062 thermo scientific nunc 24well cell culture treated multidishes 142475 step tube complexation components amount per well 24well. The development of reporter gene systems and selection methods for stable maintenance. Yous uggested to thaw a new vial of cells if the cells had reached confluency even if it was few passages before. It is used to increase the transfection efficiency of rna including mrna and sirna or plasmid dna into in vitro cell cultures by lipofection. Laboratory 2010 lipofectamine 2000 transfection protocol 10cm plates reagents. Lipofectamine 2000 protocol, with 2ul of lipofectamine 2000 per well. Add 100l of optimum to the eppendorf tube to dilute the dna and mix by tapping. Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. Does anyone have experience with the lipofectamine 2000 toxicity. I want to try to use hela cell and lipofectamine 2000 to do transfection. To transfect cells with sirna, follow the protocol as.
Lipofectamine 2000 reagent protocol 202lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. While lipofectamine 2000 reagent demonstrated transfection efficiencies only slightly lower. I use 24well plates for transfections in triplicates. Transfection of hela cells is carried out using the effectene transfection. Optimemin ul, equal volume for dna and lipofectamine lipofectamine in ul 25. Rnai or technical service, as are celltype specific rnai transfection protocols see rnai protocols. Transfection protocol transfection components component cat. Transfection efficiency in a cancer cell line panel. Data from three independent experiments performed on different days were averaged then scaled to untransfected hela controls cells alone and are represented as a percentage of the untransfected. Lipofectamine crisprmax is more robust than lipofectamine rnaimax and several other transfection reagents in delivery of cas9 proteingrna complexes into a variety of cell lines. Two days posttransfection, cells are now ready for further treatment or confocal microscopy viewing.
Reverse transfection protocol for dna in 96well plates using transitx2 dynamic delivery system. Dna complexes were added to wells so that transfection could occur as cells were attaching to the plate. On the day of transfection, prepare a single cell suspension from stock cells. Invitrogen lipofectamine 2000 transfection reagent. Plate cells so they will be 6080% confluent at the time of transfection. Being a lowefficiency method it results in very sparse distribution of transfected neurons, which is perfect for analysis of endosomes dynamics within single neurons. The nucleic acidparticle conjugates are then shot into recipient cells at a high velocity gene gun. Some of my cells will dried if i used the volume suggested by the manual. Flow cytometry hystograms of hela a and hacat b cells transfected by the lipofectamine 2000 and k2 transfection protocols. I am doing transfection using lipofectamine 2000 and plasmid dna, too. Add the lipofectaminedna mixture to each well and gently swirl to mix. Lipofectamine 2000 is cationic based lipid delivery system, which transports dna into cells by fusing with plasma membrane.
The amount of lipofectamine 2000 should be changed in proportion to the culture medium. After incubation, remove media and replace with fresh media. A comparison showing transfection efficiency of polyjet reagent vs. Livecell imaging to compare the transfection and gene. I performed lipofectamine2000 transfection on hela cells and got really good results checked transient transfection under fluorescence microscope and had very high efficiency.
Lipofectamine 3000 protocol written out with timing. Lipofectamine 2000 transfection for hek293 cells about 6070% transfection rate transfect cells 6well plate. Im about to transfect viral rna from an in vitro transcription assay by cationic liposomes, into bhk cells and i was. Twentyfour hours posttransfection the cells were harvested and cell viability assayed. Combine diluted sirnachimera and lipofectamine 2000, vortex for a few second, incubated at room temperature for 20 min, then added to a 2ml culture. Transfection of living hela cells with fluorescent poly. Transfection of fluorescently labeled oligos with lipofectamine 2000 back to table of content back to protocol and application notes. In the latter case, hela cells that stably expressed egfp were used. Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations. Hela based chart never really worked out for me to obtain 7080% for 293t, the. American medical association, manual of style, 10th edition, 2007.
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. I grow the cells in a 96 well plate until they are 80% confluent and then i follow the lipofectamine ltx for hela h3 protocol. It is critical to wash cells with one volume of dpbs and change media every day. 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 of nih3t3 cells, hela, swis 3t3, 293t with. Scaling up or down lipofectamine 3000 reagent transfections. Dna complexes were mixed with cells in suspension and the mixture was then added to the empty wells. Despite this, a satisfactory mechanismbased explanation of their. Add 6l of lipofectamine2000 from biostores and 100 l of. Reverse transfection an overview sciencedirect topics. Hela are easy to transfect and typically require less lipofectamine 2000 or 3000 vs other tougher cells. Lipofectamine 2000 dna transfection reagent protocol see page 2 to view a typical dna transfection procedure. Lipofectamine 2000 lf2k per well of a 12well plate.
With proprietary shaved cell transfection protocol, polyjet left panel gives up to 70% gfp positive cells vs. Transfect cells at high cell density for high efficiency, high expression levels, and to minimize cytotoxicity. To solve this one can titrate the amount of lipofectamine used keeping the ratio lipodna constant, limit the exposure incubate cells only for a couple of hours or so, then wash and replace with fresh medium. Improved delivery of cas9 proteingrna complexes using. Thee two most important factors in choosing transfecting reagent are. Incubate at 37c for 4 to 6 hours for delicate cells or overnight for tough cells. Can anyone provide me with a proper transfection protocol into hek293 cells with lipofectamine 2000. Even though hela cells are very hardy, the hela cell line is fairly easy to transfect and very receptive to viral vectors, making it a popular research tool in virology. 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. To optimize the transfection into hela cells by lipofectaminetm2000. Lipofectamine 2000 reagent thermo fisher scientific. Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000. Prepare plasmid dnalipid complexes recommend 2 doses of lipid. Protocol for dna transfection and luciferase assay using pfrwt plasmid.
Optimizing a lipocomplexbased gene transfer method into hela. Transfection guide overview of transfection methods promega. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. Researchers use lipofectamine 2000 reagent for sirna and shrnabased gene knockdown experiments, as well as for gene expression studies. Lipofectamine 2000 lf2000 is a reagent that i have found fits exactly what i need.
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