Choosing a transfection reagent is easier when you can rely on the work of researchers who have used it before you. Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations. We have used fugene hd in 293t cells and works very efficiently to make viruses, indeed we used it in a. According to our results, lipofectamine 3000, fugene and rnaimax. To test the transfection efficiency of the reverse transfection approach, a luciferaseencoding plasmid was reverse transfected into hek293, hep g2 and jurkat cells using fugene 6, fugene hd, transitlt1 and lipofectamine 2000 transfection reagents. 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. The authors now use the nepa21 for their transfection experiments. The fugene hd transfection reagent is lipidbased, simpletouse and can yield high transfection efficiencies with minimal cytotoxicity. Lipofectamine 2000 reagents 3t3 4 4t1 2 a431 2 a549 3 achn 2 bend. Optimized delivery of sirna into 3d tumor spheroid.
We routinely use fugene hd to introduce plasmids encoding tagged lipidbinding domains into raw264. Finally, we present a fast and efficient method for transfection of trypsinized mes cells using the liposomalbased lipofectamine 2000. Molecules free fulltext systematic screening of commonly. Comparison of transfection efficiency of nonviral gene transfer reagents. Fast and efficient transfection of mouse embryonic stem. Thus, transfection reagents differed in the extent to which they perturbed transcriptome e. Selection of transfection reagent is sometimes a puzzling issue. Description based on our proprietary polymer synthesis technology, polyjet dna in vitro transfection reagent is formulated as a. Lipofectamine 3000 reagent provided higher gfp transfection efficiency than lipofectamine 2000 and fugene hd reagents for all five cell lines.
According to our results, lipofectamine 3000, fugene and rnaimax showed high transfection efficacy, however, rnaimax may be a better option for majority of cells when lower toxicity is desired. Jun 30, 2016 finally, we present a fast and efficient method for transfection of trypsinized mes cells using the liposomalbased lipofectamine 2000. During development of each reagent, good transfection efficiency was. Lipofectamine 3000 reagent thermo fisher scientific hk.
Frequently labs try stay with their traditional technology and do not want to change it due to optimisation issue. Lipofectamine 3000 reagent is a proprietary formulation for transfecting nucleic acids into a wide range of eukaryotic cells. Optimized delivery of sirna into 3d tumor spheroid cultures. Lipofectamine 2000 reagent protocol 202 lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. Fugener hd transfection reagent technical manual tm328. Fugene hd transfection reagenta is a novel, nonliposomal formulation designed to transfect dna into a wide variety of cell lines with high efficiency and low. Effi ciency and ofp expression were analyzed 72 hours posttransfection and a u2os and b. Invitrogen lipofectamine 2000 transfection reagent. I am having very poor results when transfecting sknsh neuroblastoma cells with lipofectamine. Determination of cytotoxicity showed that the largest amount of viable cells was found after transfection with jetpei and expressfect. This study compared six commercially available reagents arrestin, expressfect, fugene hd, jetpei, lipofectamine 2000, and superfect for gene transfection. The use of fugene hd does not require culture medium change or serum removal during. Xgal staining of hepg2 cells 72 hours after transfection by fugenehd, fugene6, and competitor lipofectamine 2000, 7,000 cells well click here to view protocol fugenehd fugenehd reagent dna.
Calcium phosphate versus lipofectaminefugene transfection. The article compares four different transfection methods for human ips cells. We tested multiple transfection reagents including lipofectamine. According to our results, lipofectamine 3000, fugene and rnaimax showed high transfection efficacy, however, rnaimax may be a better option for majority of cells when lower toxicity is. Optimizing a lipocomplexbased gene transfer method into hela. Calcium phosphate versus lipofectaminefugene transfection for virus production.
American medical association, manual of style, 10th edition, 2007. Which transfection reagent is best with reporter assays on. Pdf comparison of transfection efficiency of nonviral gene. Effi ciency and ofp expression were analyzed 72 hours posttransfection and a u2os and b hepg2 cells showed 4fold and 80fold improvement with lipofectamine. To test toxicity of fugene 6, fugene hd, transitlt1 and lipofectamine 2000 with hek293, hep g2 and jurkat cells, we multiplexed our luminescent assay with a live cell viability assay. I personally have used this reagent to transfect cells for fluorescent microscopy, gst pull downs, and coimmunoprecipitation experiments.
We have used effectene, lipofectamine 2000, and fugene hd and have had the most success with fugene. Qc specifications back to table of content lipofectamine 2000 is tested for the absence of microbial contamination using. It is suitable for use in media with or without serum, and for transient or stable transfection, as well as cotransfections of multiple dna plasmids. 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. It does not contain any ingredients of human or animal origin. Promega fugene hd transfection reagent fugene hd is a novel, nonliposomal reagent for transfecting dna into a variety of cell lines with high efficiency and low. Vortex and incubate the mixture at room temperature for 15 min before adding to the well. We have used fugene hd in 293t cells and works very efficiently to make viruses, indeed we used it in a mini. Fugene hd transfection reagent is a multicomponent reagent that forms a complex with dna, then transports the complex into animal or insect cells. Fugenehd in volume of 4l along with 105 hela cells, transfection. Laboratory 2010 lipofectamine 2000 transfection protocol 10cm plates reagents. Which transfection reagent is best with reporter assays on stem cells. While lipofectamine 2000 reagent demonstrated transfection efficiencies only slightly lower those of viafect reagent, it was considerably more toxic to hela cells at all tested ratios of reagent to dna.
Luciferase activity was measured after 24 hours figures 24. The ratio of transfection reagent to episomal vectors was varied, and the. Optimizing a lipocomplexbased gene transfer method into. The lipofectamine ltx and fugene hd lipidbased transfection reagents were used in combination to transfect primary fibro blasts and hepatoblasts, showing. Delivery of episomal vectors into primary cells by means of. An example showing optimization of transfection conditions for the fugene hd reagent in a 96well plate is shown in figure 2. I tested several transfection reagents at the same time fugene hd, mirus. What this product does number of transfection experiments in a typical experiment using hela or cos1 cells, 1 ml of fugene hd transfection reagent can be used to perform up to three hundred transfections in 35mm tissueculture dishes, using 3 general laboratory equipmentl of reagent.
I chose fugene hd according to the advices from gan jen yang and ahmet cingoz. Fugene hd transfection reagent is a proprietary, nonliposomal, low toxicity formulation designed for high efficiency delivery of dna into a broad range of cell lines. Using fugenehd in volume of 4l along with 105 hela cells, transfection efficiency. Maximal transfection efficiency in mc3t3e1, c3h10t12, hela, c2c12, hep g2, and hct116 was seen using fugene hd, in pt30, 4t1, and hekn was seen using arrestin, and in mcf7 was seen using jetpei.
Thank you so muchmcri tried both of them and lipofectamine 2000 was much betterin 293ft cells. The lipofectamine 3000 is the bees knees though, i managed to get loads. I use 24well plates for transfections in triplicates. Nonviral genetic transfection of rat schwann cells with. Make dnalipofectamine 3000 complexes in serumfree medium such as.
Nonviral genetic transfection of rat schwann cells with fugene hd lipofection and amaxa nucleofection is feasible but impairs cell viability volume 6 issue 4 armin. Fugene hd transfection reagent for efficient mammalian cell. Researchers use lipofectamine 2000 reagent for sirna and shrnabased gene knockdown experiments, as well as for gene expression studies. Invitrogen lipofectamine 2000 transfection reagent fisher. Xtremegene 9 dna transfection reagent is a proprietary blend of lipids and other components supplied in 80% ethanol, filtered through 0. Fugene hd, jetpei, lipofectamine 2000, and superfect for gene transfection. All three offer broad spectrum transfection capability and low toxicity but the same question always comes up. Frequently labs try stay with their traditional technology and do. Xtremegene 9 dna transfection reagent sigmaaldrich. Fugene hd produced the fewest total number of differentially expressed transcripts.
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. Comparison of transfection efficiency of nonviral gene. Lipofectamine 2000 affected the largest number of transcripts, followed by effectene and then lipofectamine ltx with plus reagent. Pdf this study compared six commercially available reagents arrestin, expressfect, fugene hd, jetpei, lipofectamine 2000, and. Promega fugene hd transfection reagent fugene hd is a novel, nonliposomal reagent for transfecting dna into a variety of cell lines with high efficiency and low toxicity. Always mix fugene 6 reagent prior to use vortex for one second or use inversion. Lipofectamine 3000 reagent poster thermo fisher scientific. Description based on our proprietary polymer synthesis technology, polyjet dna in vitro transfection reagent is formulated as a biodegradable polymer based dna transfection reagent that ensures effective and reproducible transfection on hek293, cos7, nih3t3, hela, cho and a broad ranges of hardtotransfect mammalian cells.
The protocol does not require removal of serum or culture medium and does not require washing or changing of medium after introducing the reagentdna complex. In this article about talen similar to zinc finger nuclease, the authors say that the nepa21 is better than the neon device. The authors now use the nepa21 for their transfection. Fugene hd transfection reagent for efficient mammalian. Fast and efficient transfection of mouse embryonic stem cells. We examined the efficiency and cytotoxicity using nine different cell lines mc3t3e1 mouse preosteoblasts, pt30 human epithelial precancer cells, c3h10t12 mouse stem cells, mcf7 human. Xgal staining of hepg2 cells after transfection by fugenehd. 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 reagent protocol 202lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart.
Each reagent was tested with a range of transfection reagent. The transfection efficacy of lipofectamine 2000 was compromised by its high toxicity, which may adversely affect its application in most cells. Calcium phosphate method vs lipofectamine tissue and cell. With only a fiveminute long transfection time we obtained at least 85 % transfected cells with 80 % maintained viability. Lipofectamine 2000 transfection reagent from invitrogen. Allow fugene hd transfection reagent to reach room temperature, and mix. Lipofectamine 2000 reagent fugene hd reagent 120% 10 nm 30 nm 80% 20% n e.
The fugene hd transfection reagent is a nonliposomal formulation designed to transfect dna into a wide variety of cell lines with high efficiency and low toxicity. Qc specifications back to table of content lipofectamine 2000 is tested for the absence of microbial contamination using blood agar plates, sabaraud dextrose agar plates, and fluid thioglycolate medium, and functionally by transfection of chok1 cells with a reporter plasmid. Fugene 6 transfection reagent is a nonliposomal formulation designed to transfect plasmid dna into a wide variety of cell lines with high efficiency and low toxicity. Lipofectamine 3000 reagent is a proprietary formulation for transfecting nucleic acids into a wide range of eukaryotic cells and especially designed for hard to transfect cells. The celltiterfluor reagent measures viability of cells by detecting the production of a fluorophore through livecell protease activity 2. The transfection protocol for lipofectamine 3000 was developed to be easy to use while still ensuring optimum performance and reliability in a wide panel of cell lines. For additional information on scaling your transfection reaction, see page 4. Theres a difference between the two protocols lipofectamine 3000 has an extra reagent that needs to be added. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. Lipofectamine reagents go online to view related products.
Table 2 examines transfection efficiency and cell viability under optimal conditions for each reagent 24 hours after transfection. Lipofectamine 2000 reagent is a lipidderived reagent designed to deliver exogenic nucleic acids dna andor rna into eukaryotic cells, either adherent or in. Comparing viafect, fugene and lipofectamine performance. Helen sarantis, sergio grinstein, in methods in cell biology, 2012. I dont know if it should be better to try the calcium phosphate method, as i would need a massive amount of plasmid. Lipofectamine 3000 reagent thermo fisher scientific kr.
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