Transfection of hek293 cells with lipofectamine 2000 manual pdf

Transient transfection of glosensor camp hek293 cells. So i did the tranfection, followed up by western blotting. One day prior to transfection, adherent cells were plated onto 24well plates at 0. Hi, i tried to transfect my hek293t cells with pegfp using lipofectamine 3000, following manufacturers instructions. The transfection permissible hek293ft cell line was used as a control in transfection procedure. Jul 19, 2016 we use cationic lipidbased transfection reagents felgner and ringold, 1989, lipofectamine 2000, lipofectamine 3000, and nonliposomal fugene hd formulation to transiently transfect the 293ft cells with 2. Combine diluted sirnachimera and lipofectamine 2000, vortex for a few second, incubated at room temperature for 20 min, then added to a 2ml culture. Here are possible causes for reduced viability following transfection, along with suggested solutions. Lipofectamine 3000 reagent outperforms invitrogen lipofectamine 2000 and fugene hd reagents. Lipofectamine 3000 reagent thermo fisher scientific sa. I do have cell toxicity, but not so serious as yours. Plate cells so they will be 7090% confluent at the time of transfection. Please refer to the product datasheet for complete protocol details.

Following transfection the cells were incubated at 37c in humidifiedair 5% co 2 for 2 h. To transfect cells in different tissue culture formats, vary the amounts of lipofectamine 2000, nucleic acid, cells, and. 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. This has made them a popular research tool in cell biology studies and an ideal cell line for therapeutic. Oligo into mammalian cells using lipofectamine 2000. General procedure for transfection of mammalian cells with. Lipofectamine 2000 reagent protocol thermo fisher scientific. You can not use antibiotics during transfection step because it will cause cell death. Cell transfection in a 24well plate using crisprmax. Cotransfection protocol for delivery of multiple plasmid dnas in a 6well plate with. Hilden, germany according manufacturers instructions and the dna was. The amount of lipofectamine 2000 should be changed in proportion to the culture medium.

Component 96well 24well 6well final dna per well 100 ng 500 ng 2500 ng. 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. I used it with different cancer cell lines and worked well. Transfection p19 cells with lipofectamine 2000 the same protocol can be used for 293t cells 1. The trusted, simple, broadspectrum transfection reagent for most cell lines. The lenti293t is an hek 293 derived cell line stably expresses the sv40 large t antigen and neomycin resistance gene. Altogen biosystems provides in vivo transfection reagents, over 100 preoptimized in vitro transfection kits for cell lines and primary cells, and electroporation delivery products. After concentrating cells to such high densities, transfection of hek293 cells becomes possible in most commonly used media and is not restricted to a specific medium. Once cells have been transfected, various methods can be used for analysis post transfection and for assessing transfection efficiency. Invitrogen lipofectamine 2000 transfection reagent is a versatile transfection reagent that has been shown to effectively transfect a variety of payloads into the widest variety of adherent.

Original article an improved method for increasing the efficiency of. Transfection efficiency is best if hek293 cells are maintained by splitting cells every 3 days note 2 and 3. Serumfree hek293 transfection media contains components optimized for cell transfection. Analyze the cells whichever way you need to for whatever it is you transfected them with. This reagent delivers superior transfection efficiency and improved cell viability for the widest range of hardtotransfect and common cells e. Cells need to double with a 24 hr doubling rate to be ready for transfection d. Successfully transfect the widest variety of biologically relevant cell types with a reagent that offers.

Cellrelated factors affecting transfection include cell. Cells for transfection were harvested at approximately 1,000,000 cells ml. 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. In previous, i performed the transfection in hek293 using lipofectamine 2000 invitrogen and followed the protocol of there for 12 well vessel. Expi293 expression system user guide for scalable transfection of expi293f cell lines in a chemically defined, serumfree medium, using the expifectamine 293 transfection kit for scalable metabolic protein labeling using expi293f cell lines, in a chemically defined, serumfree, methioninefree medium using a.

Transient transfection of mammalian cells with lipofectamine. Plasmid dna and lipofectamine 2000 invitrogen, carlsbad, ca were. Each reagent was used to transfect hek 293, hela, lncap, hepg2, and a549 cell lines in a 96well format, and gfp expression was analyzed 48 hours posttransfection. Cas9 protein and hprt1 grna were transfected into hek293 and hct116 cell lines according to manufacturers protocol. Add dnalipofectamine complexes 500 ul to the 2 ml of media on the cells. Approximately 1824 hours before transfection, plate cells in 2. I am trying to optimize the transfection by using lipofectamine 2000 for 2000 ng of plasmid. We have compared transfecting cells in media with and without antibiotics in multiple cell lines, assessed both the transfection efficiency and toxicity, and found no difference. Lipofectamine 2000 dna transfection reagent protocol see page 2 to view a typical dna transfection procedure.

Lipofectamine and nucleofection of hek 293 were essentially equivalent in terms of toxicity and efficiency. Hek2936e cell preparation for transfection 20101121jv. The microscopic analysis was performed using antirigi mab. All of the transit broadspectrum and celltype specific dna transfection reagents can. This allows the dna and the lipofectamine to form the complexes that will get the dna into the cells. Add 6l of lipofectamine2000 from biostores and 100 l of. Prewarm 50ml of optimum stored in cold room at 4c 10min. Higher densities can lead to spontaneous differentiation. Liposome lipidbased formulation has the least influence on. Lipofectamine ltx dna transfection reagent protocol see page 2 to view a typical plasmid transfection procedure. Divide the cells 1824 hours before transfection to ensure that the cells are actively dividing and reach the. Lipidbased transfectiontroubleshooting thermo fisher. Htm cells were transfected using lipofectamine with a fluorescent mirna dy547 and analyzed 48 hours after transfection.

However, unlike fugene and lipofectamine 3000 that displayed strong toxicity to hek293 cells 65. 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. When splitting the cells, dilute them to no less than 1x10e6. Perform reverse transfection by preparing complexes inside the wells, and then adding cells and medium. Transfection of human hek 293f cells with metafectene pro. 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. On day 1, glosensor camp hek293 cells were split into 10cm tissue culture dishes at a density sufficient to achieve 50% confluence by day 2. I am doing transfection using lipofectamine 2000 and plasmid dna, too. 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. To transfect cells with sirna, follow the protocol as. Lipofectamine 2000 transfection reagent from invitrogen. My main purpose is to show that my antibody for bob works.

Cells were seeded in 96well flat bottom nontissue culture plate just before transfection with the density 50,000 cells per well in 100. After 4h aspirate media and refeed cells with normal media. Optimized conditions for successful transfection of human. Add dna lipofectamine complexes 500 ul to the 2 ml of media on the cells. Glosensor camp hek293 cells were transiently transfected with wildtype or h223r hpth 1 r expression plasmids on day 2. Lipofectamine 2000 cd reagent is a proprietary animal originfree formulation for transfecting nucleic acids into eukaryotic cells. Transfection reagents are highly efficient for dna and sirna transfection in vivo and in vitro. The best transfection reagent and conditions for a particular cell type must be empirically and systematically. Dna lipofectamine 2000 complexes can be added directly to cells in culture medium. The recommended cell density for hek 293 at transfection is. One is pcdna3 based protein a and the other is pcdna5frtto based gfptagged protein b. Altogen cro offers in vivo rnai services, tumor xenograft models, toxicology testing, stable cell line generation, and.

Please note that as per recent findings, antibiotics can be used in media during transfection. Mouse myeloma cell lines sp20, ns0, ns1, ag8, and p3u1 were transfected with pegfpn1 vector using lipofectamine 2000, jetpei and lyovec commercial transfection reagents in different combinations. Transfection of cells with different lipofectamine 2000 amounts. Transfection conditions for expi293f cells for each 30ml transfection, use 7. Expi293 expression system thermo fisher scientific. Scale your transfections up or down by proportionately adjusting the amounts of the reagents used. Several cell densities and lipid volumes were investigated to determine optimal transfection conditions, shown by the shaded boxes. For adherent cells, we recommend 9095% confluence at the time of transfection for high efficiency, high expression levels, and to minimize cytotoxicity. 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. I want to do a transfection to overexpress protein bob in 293t cells. A guide to transient expression of membrane proteins in hek. Hek293 transfection reagent is tested functionally by transfection of hek293 cells with a small. A hela cells were transfected with hcv dsrna using lipofectamine 2000 reagent. Dnalipofectamine 2000 complexes can be added directly to cells in culture medium.

Determine the optimal cell density for each hek 293 cell subtype in order to maximize transfection efficiency. What i found is lipofectamine 2000 is toxic to cells, so i reduced the recommended quantity of the invitrogen protocol. Co transfection of 2 plasmids in hek293 cells i am trying to cotransfect 2 plasmids in hek293 cells. Three cell densities of hepg2 cells were transfected with gapd sirna 100 nm using a range of volumes 0. With the exception of hek 293 cell transfection, nucleofection proved to be less toxic and more efficient at effectively delivering dna into the cells as determined by cell proliferation and gfp expression, respectively.

A guide to transient expression of membrane proteins in. Take care to maintain a standard seeding protocol between experiments. Highest transfection efficiency in many cell types and formats e. Prepare plasmid dnalipid complexes recommend 2 doses of lipid. Thus, hek293 is an excellent cell line to use in transfection experiments or to produce recombinant dna or gene products. Lipofectamine 2000 dna transfection reagent protocol.

The viafect transfection reagent from promega is a very good for transfecting many different cell types with low toxicity. Lower confluence will achieve higher percentage of transfection but may yield a lower absolute number of transfected cells. I want to try to use hela cell and lipofectamine 2000 to do. For suspension cultures, transfect cells at a density of 0. Scaling up or down lipofectamine 3000 reagent transfections use the following table to scale the volumes for your transfection experiment. Ideally have the cells double two consecutive times at maximum doubling rate log phase before transfection e. Gently mix media on cells via figure 8 pattern and back and forth motions. L reverse transfection of rnai reverse transfection is faster to perform than forward transfection and is the method of choice for highthroughput transfection. Can anyone provide me with a proper transfection protocol. This is necessary because serum is replete with rnases that degrade. Stealthsirna transfection protocol lipofectamine 2000.

Prior to transfection, hek 293 cells are preincubated with serumfree dmem medium for 1 h. Lipofectamine transfection of c2c12 cells bridges lab. Comparative transfection of dna into primary and transformed. Thee two most important factors in choosing transfecting reagent are. For additional information on scaling your transfection reaction, see page 4.

Optimization of gene transfection in murine myeloma cell. We improved a process for transient calcium phosphatebased transfection of hek293 ebna cells in a 1. Transfection amounts component 96well 24well 6well dna per well 100 ng 500 ng 2500 ng plus reagent per well 0. Grow cells at least until they are 2x10e6 but no denser than 3x10e6. Lipofectamine 2000 transfection for hek293 cells about 6070% transfection rate transfect cells 6well plate. Hek293 cells exhibited the highest egfp expression compared to the bj fibroblasts and ecs. Add 100l of optimum to the eppendorf tube to dilute the dna and mix by tapping. Invitrogen lipofectamine 3000 transfection reagent. With the exception of hek 293 cell transfection, nucleofection proved to be less toxic and. Wash the cells 2x pbs seed 10 6 cells in 300 ul warm optimem in 24 wellsplates. Calculating stable transfection efficiency 12 viii.

Youll wait 1 to 3 days after transfection before analyzing the cells. Take care to maintain a standard seeding protocol between experi ments. Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. Lipofectamine 2000 for transfection provides the following advantages. Improved delivery of cas9 proteingrna complexes using. Transfection amounts component 96well 24well 6well dna per well 100 ng 500 ng 2500 ng plus reagent optional 0. Materials needed have the following reagents on hand before beginning. Expression in the presence of various transfection reagents a hela cells were treated with different transfection reagents for 6 hr without plasmids, according to the companys manual. There are many factors that can influence transfection efficiency, a number of which are specific to the target cell. Good news, my antibody works pretty well tested 293t untransfected, transfected 293 two different clones, and psirpg transfected cells transfection control. 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. Lipofectamine 3000 protocol written out with timing. With automated, highthroughput systems, a complexing volume of 50 l is recommended for transfections in 96well plates.

Transfection of nih3t3 cells, hela, swis 3t3, 293t with. However, i could only reach at most 2030% transfection efficiency. For each transfection sample, prepare dnalipofectamine 2000 complexes as follows. Lipofectamine 3000, fugene and rnaimax could be used for screening sso in hek293 cells. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. To transfect cells with sirna, follow the protocol as described for dna but.

Plate cells for 212hrs but not over 24 hours prior to transfection to achieve a 4080% confluence at the time of transfection. I always use lipofectamine 2000 for transfection of hek293t cells. The reagent provides high transfection efficiency in many cells types and formats. Nov 20, 2008 in my experience lipofectamine works like a charm in many cells, including hek293. Lipofectamine 2000 cd reagent complexes can be added directly to cells in culture medium. If you let them reach confluency, even if this happened a few passages ago, the transfection efficiency will be significantly diminished. Hek293 cells are easy to grow in culture and transfection kits are commercially available.

Scaling up or down lipofectamine 3000 reagent transfections. Mar 03, 2014 the transfection of cells already with 0. I use 24well plates for transfections in triplicates. Maintain p19 cells undifferentiated in mem with 10% serum 7.

Hek293 transfection reagent is supplied in liquid form at a concentration of 0. Then, cells were washed with pbs twice and transduced with scaav2cbapegfp at 2,000 vgs per cell. Optimizing transfection lipofectamine 2000 cd reagent. A table listing recommended blockit fluorescent oligo and lipofectamine 2000 amounts and volumes to use for transfection in different tissue culture formats.

Transfection commonly refers to the introduction of nucleic acids into eukaryotic cells, or more specifically, into animal cells. The transfection medium was then removed and the cells were incubated for an additional 48 h in complete medium 2 ml per well. For other tissue culture formats, refer to the user protocol pdf. I agree with parvoman that one of the main considerations is how well the cells are growing. Hek 293 cells are efficiently transfected with lipofectamine, with typically 70% of cells showing fluorescence following transfection with green fluorescent protein gfp. Cotransfection protocol for multiple plasmid dnas mirus bio. Each transfection was carried out in triplicate and repeated 2 to 3 times.