Minimizing reagentdependent side effects as analyzed by geneexpression profiling and cytotoxicity assays. Request pdf comparing reagents for efficient transfection of human primary. Citation list of established cells transfected with fugene 6 transfection reagent 10t12 timothy a. Vwr offers transfection reagents for numerous applications, including bioproduction, in vivo work, viral production, dna and sirna transfection, crisprcas 9 and more. For use with application transfect dna into a wide variety of cell lines with high efficiency and low toxicity, for use with luciferase. Fugene 6 transfection reagent from roche applied science. After 18h of incubation, medium was replaced with high serum growth medium 30% fcs vv. This protocol is used to generate baculoviruses containing gpcr genes of interest for expression in sf9 cells. Fugene 6 transfection reagent has been commercially available since 1997. 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. This protocol is a slight modification of expression systems protocols for generating viruses.
The polarized organization of neurons depends on the selective targeting of cargoes to either axons or dendrites, driven by motor proteins that move selectively toward either the plus or the minus end of microtubules bentley and banker, 2016, britt et al. Citation list of established cells transfected with fugene. Transfection is the process of deliberately introducing naked or purified nucleic acids into eukaryotic cells. Supporting online material for whitehead institute. Roche fugene 6 transfection reagent is a nonliposomal multicomponent reagent wellsuited for experiments involving cellular analysis. Use the table below to find the right product for your experiment. This file contains supplementary figures 110 and supplementary. Production of retroviruses using fugene6 32100 1 on the day before transfection, plate 0.
Virus titer was determined on lowpassagenumber tzmbl cells by. Addgene is working with the trc to make this shrna cloning vector available to the scientific community. Fugene 6 transfection reagent university of pittsburgh. Fugene 6 transfection reagent multicomponent formulation for the transfection of eukaryotic cells cat. Fugene transfection protocol 1 add 3ul of fugene to 97ul of optimem, being sure to avoid touching the pipette tip to the side of the tube as the fugene will stick to plastic. The constructs were transfected into a helpervirus free amphotropic producer cell line phoenixa, a derivative of the human embryonic kidney cell line 293 a kind gift of dr g.
On day five, cells were rinsed once with amino acidfree schneiders. Differentiation between oppositely oriented microtubules. Cells must be healthy and free of mycoplasma and other contaminants. Fugene 6, one of fugents premier products, is a multicomponent lipidbased transfection reagent. Lif you have used fugene 6 transfection reagent in the past, we suggest that you increase the plating density for initial tests using fugene hd transfection reagent. Fugene 6 transfection reagenta is a nonliposomal reagent that transfects dna into a wide variety of cell lines with high e.
We use roche xtreme gene 9 which is the next gen for fugene. Fugene is a transfection reagent that apparently increases transfection rates for animal cells. Always dilute fugene 6 transfection reagent directly into medium without contacting the side of the tube. And i mix 200ul optimem with 12ul fugene 6 without touching the tube. Incubate the fugenehd transfection reagentdna mixture for 015 minutes at room temperature.
Lipofectamine2000 works quite well in 293cells 90% confluent in my. Nanog overexpression allows human mesenchymal stem cells. Rna helicase ddx3 is a regulatory subunit of casein kinase 1 in wnt. Comparing reagents for efficient transfection of human primary. Fugene 6 transfection reagent is stabilized for extended storage. Sigma aldrich, using fugene 6 roche, berlin, germany in optimem medium gibco. For details of the experiments described above, see biochemica 4, 911 2004, available online. Just to be sure dna is not an issue here, try to precipitate dna and transfect it. Fugene 6 reagent complex prior to plating, making it a strong candidate for high throughput applications. Intraperitoneal gene delivery mediated by a novel cationic.
On day 1, 4 million s2 cells were plated in 6cm culture dishes in 5 ml of express five sfm media. It may also refer to other methods and cell types, although other terms are often preferred. Both fugene 6 and fugene hd were able to transfect a luciferasecontaining plasmid using a reverse transfection protocol resulting in high luminescence signal. Storage and stability fugene 6 reagent is shipped at room temperature. Two days later, a second round of dsrna transfection was performed. Rock inhibition enhances airway epithelial basal cell.
Nanog overexpression allows human mesenchymal stem cells to. Positive clones were validated with microscopy, flow. Cells were transfected with 1 g of dsrna per million cells using fugene 6 roche. Xtremegene transfection reagents from roche have been used for a wide range of applications and a vast array of cell lines, primary cells and stem cells. Since that time, its popularity has increased due to its ease of use, minimal to no cytotoxicity, and the high level of transfection in many different cell lines. After 48 h, cells were washed in 1x pbs and pelleted. The gentle performance of the fugene 6 transfection reagent makes it an.
Description 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. Supplementary appendix this appendix has been provided by the authors to give readers additional information about their work. The protocol does not require removal of serum or culture medium and does not require washing or changing of medium. Viruses were harvested at 48h and 72h posttransfection.
On day one, 4,000,000 s2 cells were plated in 6 cm culture dishes in 5 ml of express five sfm media. The cells are ready for transfection after 1820 hours, or when they are about 2530% confluent. After 48 hours, cells were subjected to positive selection using 1. Product fugene 6 transfection reagent, 1ml product code e2691 substance number description amount symbols e269 fugene 6 transfection reagent 1 f.
The plasmid contains a cloning site for the insertion of shrna. 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. Cos7 cells were transfected at 80% confluency using fugene hd roche. Formulation fugene 6 transfection reagent is a proprietary blend of lipids and other components supplied in 80% ethanol, sterilefiltered, and packaged in glass vials. Clustered cells that resembled nanogtransfected hmscs were passed by mechanical dissociation from the. Additionally, low cell numbers can be transfected in 96well plates.
General considerations successful transfection involves optimizing the fugene 6 transfection reagent. Avoid contact of undiluted fugenehd transfection reagent with the sides of the tube or plate. Figure 3 transfection activity of lipofectin, lipofectamine, fugene 6. Production of retroviruses using fugene 6 32100 1 on the day before transfection, plate 0. Hela cells were cotransfected using fugene 6 roche with pcmvpuromycin and gpr68egfp in a 1. The lipid fugene 6 roche, mannheim, germany was employed as gene. Plate slightly more cells when making vsvg pseudotyped viruses. Fugene 6 transfection reagent is gentle on the cells. Nolan using either calcium phosphate life technologies bv, breda, the netherlands or fugene6 roche diagnostics, according to manufacturers protocols. Fugene 6 roche, indianapolis, in as previously described 5, 6. Catenin signaling cristinamaria cruciat, christine dolde, reinoud e. Transfection of sf9 cells in suspension december, 2008 version 1. Fugene 6 transfection reagent allows for the efficient delivery of dna vectors from very large plasmids to oligonucleotides and nucleotides into a broad range of cell types with extremely low cytotoxicity. Figure 1 vertiga shaker manufactured by thomson instruments co.
Fugene 6 transfection reagent is a proprietary blend of lipids and other components supplied in 80% ethanol, sterilefiltered, and packaged in glass vials. We use it in lab for cell culture transfections, but i have no idea what it is or how it works. Korswagen, christof niehrs to whom correspondence should be addressed. Virus titer was determined on lowpassagenumber tzmbl cells by xgal staining as readout. Lofan sirnafugene 6 mixture was added, and the cells were incubated for 48 h in a 5%co 2. The choice of transfection reagent depends on the cell type used. The reporter plasmids and the expression plasmids were transfected into nih3t3 cells using fugene 6 roche applied science. Longmore, washington university school of medicine, 7.
Gag aug auc ugg aac3 into cells using fugene 6 roche, mannheim, germany according to the manufacturers instructions. Nfatcontrolled expression of gfp permits visualization. For low ph experiments 5mm hepes and mes were added to the media and ph adjusted with hcl. Mirus bio files lawsuit alleging that roches fugene 6. The yfp reporter gene expressed by the virus was subcloned into the pfu plasmid that also included a puromycin resistance gene kindly provided by g. The new custompack of fugene 6 transfection reagent. Mirus bio files lawsuit alleging that roche s fugene 6 transfection reagent infringes patent on september 12, 2011, mirus bio sued fugent, llc for allegedly infringing its patent entitled process of transfecting a cell with a polynucleotide mixed with an amphipathic compound and a dnabinding protein u. Supplemental materials and methods cell culture necs were isolated and cultured as previously described 1. Lentiviral infection of shrnas or orfs was performed as described 7, 15. For selection of virally infected cells, 12 gml of puromycin plko. The lentiviral construct for the generation of ahr reporter cell lines was obtained from sabiosciences.
Fugene 6 transfection reagent is a nonliposomal reagent that transfects dna into a wide variety of cell lines with high efficiency and low toxicity. Adherent cells can be trypsinized and transfected by the dna. Of course, efficiency will be lower but having too much dna could alter transfection efficiency. Fugene 6 transfection reagent technical manualpdf 811 kb english. Due to the simple methodology of fugene 6 transfection reagent, minimal optimization, low cytotoxicity, and ability to provide high transfection efficiency even in the presence of serum, it is one of the. Proliferative clusters began to emerge after one week and grew large enough for expansion typically by three weeks. Thank you so muchmcri tried both of them and lipofectamine 2000 was much betterin 293ft cells.
Olson 2000 activation of the myocyte enhancer factor2 transcription factor by calciumcalmodulindependent protein kinasestimulated binding of 1433 to histone deacetylase 5. Briefly, 14 million cv1 cells 80% confluency, t175 cm 2 flask were transfected with a 1. Transfection of sf9 cells in suspension december, 2008. Fugene 6 transfection reagent with plastic surfaces. Three days later, cells were harvested, counted, and plated at a density of 5. In animal cells, transfection is the preferred term as. Citation list of established cells transfected with fugene 6. 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. Transfection of sf9 cells in suspension nih common fund.
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