Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. Xtremegene hp dna transfection reagent highperformance. Add 100l of optimum to the eppendorf tube to dilute the dna and mix by tapping. Rna was 20 times diluted in optimemi reduced serum medium invitrogen and transfected into raw 264.
Dna, sirna, mirna, mrna, viral rna, crisprcas9 components, and oligonucleotides to. In addition, if the lentiviral transfection efficiency is truly low, which is the case for raw264. Lipofectamine 2000 transfection reagent dartmouth college. Lipofectamine 2000 transfection, the expression of gfp was detected in 76,4% of hela cells with a mean fluorescence intensity of 437. Detection of murine norovirus 1 by using plaque assay. Im having problems transfecting my raw cells using lipofectamine or lipofectamine ltx. Our reagents are ideal for delivering multiple types of nucleic acids including.
Preparation of the cells for transfection the day before transfection. Whole field 20x magnification view of gfp expression in raw 264. Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations. Although these cells are quite useful for studying innate immune. Transfection efficiencies lower than 50% were not reported. In hacat cells, lipofectamine 2000 achieved a transfection efficiency of 22. Cells are transfected with sirna twice on subsequent days.
Targefect raw is a new reagent from targeting systems specifically designed for the transfection of the raw 264. Altogen biosystems provides in vivo transfection reagents, over 100 preoptimized in vitro transfection kits for cell lines and primary cells, and electroporation delivery products. Transient plasmid dna transfection into hela and hacat cells. A method for the establishment of a cell line with stable expression. The human monocytic cell line thp1 has been shown to express sting and respond to 23. Lipofectamine 2000 transfection reagent is versatile transfection reagent that has been shown to effectively transfect widest variety of adherent and suspension cell lines. One approach to this technique is transient transfection of genespecific small interfering rnas sirnas. They were growing fine until recently i see that while growing, the cells reach confluence but there. Transfection transit transfection reagents mirus bio. Transfection into the macrophage cell line, raw264. Invitrogen lipofectamine 2000 transfection reagent fisher. The following protocol describes how to transfect plasmid dna into raw 264. Examples of cells transfected successfully thermo fisher.
A polymerbased transfection caco2 kit for colon adenocarcinoma cells yields at least 94% transfection. For some cell types, such as cos 7, the transfection efficiency was over 95% while other hardertotransfect cells, such as caco2, displayed efficiencies around 75%. A typical forward transfection protocol using transit2020 transfection reagent can be found here. Transfection efficiency was assessed by facs analysis in various cell lines 24 h after transfection in 24well plates. The reagent is compatible with various media formulations. Theres a difference between the two protocols lipofectamine 3000 has an extra reagent that needs to be added. Can anyone recommend the most efficient transfection method. The following procedure is for simultaneous transfection and plating of raw 264. Compatibility with serumcontaining media low cytotoxicity. Lipofectamine ltx reagent is a proprietary, animalorigin free formulation for the transfection of dna into eukaryotic cells with low cytotoxicity.
For suspension cells andor high throughput applications, a reverse transfection protocol where freshly passaged cells are added to preplated transfection complexes is ideal as it reduces handson time for the end user. Transfection reagent lipofectamine triggers type i. I already increase the puromycin as 16 ugml, on the 4th day of antibiotic added, still has 10%. Altogen biosystems provides efficient biodegradable transfection reagent kits for the caco2 colon cancer cell line. In biontex experiments gfp positivity increased to 90. Afcs procedure protocol pp00000182 version 1, 103103. Lipofectamine 3000 reagent transfection efficiency fold protein expression improvement, lipofectamine 3000 vs 2000 reagent cell type origin. Lipofectamine 3000 reagent thermo fisher scientific sa. Lipofectamine 2000 reagent thermo fisher scientific. Add 6l of lipofectamine2000 from biostores and 100 l of.
To examine the effect of lipofectamine on host cell immune response, raw 264. Can anyone recommend the most efficient transfection. Hela cells, near confluent sixwell plates were transfected with. Choosing the right transfection reagent for optimal efficiency. 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. Innate immune responses of primary murine macrophagelineage cells and raw 264. Read 17 answers by scientists with 10 recommendations from their colleagues to the question asked by jennifer gilbert on jun 28, 20. 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. Targefectraw from targeting systems biocompare product.
Lipopolysaccharideinduced mir1224 negatively regulates. In tube b place 190 l of optimemi and 10 l lipofectamine 2000. Use a range of several ul of lipofectamine and ug dna to increase viability after transfection and efficiency. Automated highthroughput sirna transfection in raw 264. We support multiple transfection methods with a different reagent for nearly any application. China, according to the manufacturers instructions, suspended in. Transfection of dna by lipofectamine in cells has been used as a powerful tool for identification of the biological function of genes. Arpe19 transfection kit retinal pigment epithelium. I use 24well plates for transfections in triplicates. Genexplus transfection of plasmid dna into raw 264.
Invitrogen lipofectamine 3000 transfection reagent fisher. The weight ratio of lipofectamine 2000 to pdna was kept constant at 3. The targefectraw reagent is combined with the virofect enhancer for optimal transfection of raw 264. Xtremegene hp dna transfection reagent is a high performance reagent that transfects a broad range of eukaryotic cells, including insect cells and hardtotransfect cell lines e. Transfection efficiency in hardtotransfect raw 264. School of biological sciences, university of southampton, bassett crescent east, southampton, hants.
Sixteen hours after transfection, lps was added to the medium at a final concentration of 100 ngml per well. To perform transfection experiments in other cell culture plates, simply multiply the suggested quantities by the relative surface area of your plate. Lipofectamine 2000 procedure for simultaneously transfecting. Transfection of nih3t3 cells, hela, swis 3t3, 293t with. Mdamb transfection kit breast adenocarcinoma altogen. Researchers use lipofectamine 2000 reagent for sirna and shrnabased gene knockdown experiments, as well as for gene expression studies. 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. Do you know any protocol for transfection of raw macrophages. Mix optimem and lipofectamine in another eppendorf 32 l lipofectamine in 0,8 ml optimem 3. Oz biosciences developped transfection reagents since almost 15 years please refer to the link below and we have reagents that are really efficient for raw 264. Macrophages occupy a pivotal position in regulating numerous aspects of the immune response. Lipofectamine 2000 transfection reagent from invitrogen. A sample protocol is listed here for transfection experiments performed in 6well plates.
Do you know any protocol for transfection of raw264. 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. Pool dishes of cells that have been prepared for transfection. The following procedure involves the transfection of sirna into raw 264.
The next day, 24g of pmeluc cdna a cdna engineered in our laboratory and 40 l of metafectenepro figureme 2amountthe ofsa lipofectamine 2000 invitrogen. In this protocol, we present an optimized transfection procedure to introduce luciferase reporter genes as a plasmid dna into the raw264. Lipofectamine 3000 reagent is designed to efficiently transfect difficulttotransfect cells, yielding superior transfection performance across the broadest array of cell types. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. Genscript recommends using lipofectamine 2000 for all transfections. For cellfectin dmriec, and lipofectamine 2000 reagents this is normal. Mix optimem with dna in an eppendorf 510 g dna0,8 ml optimem 2. This reference provides a recommended procedure to transfect plasmid dna into raw264.
Complexes were formed for 5 minutes prior to applying 5l of the complex mixture to 50,000 cellswell in a 96well plate. Genmute transfection buffer 5x, formulated for maximal transfection efficiency, 8. Viafect reagent provided optimal transfection efficiency and low toxicity for the majority of the cell lines tested, although there were some cell lines e. We described here a gentle yet robust procedure for transfecting luciferase reporter genes into raw264. 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.
In tube a place 2 g of dna in optimemi for a final volume of 200 l. Highest transfection efficiency in many cell types and formats e. Fibroblast 3t3, cos 7 myoblast c2c12, l6 crl1458 hepatocyte hepg2, huh7 erythroleukemic cell k562. Lipofectamine 2000 reagent protocol 202lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. A case study for optimization procedure jeanphilippe carralot, 1 taekyu kim, boris lenseigne,2 annette. Transfection reagents are highly efficient for dna and sirna transfection. To transfect cells with sirna, follow the protocol as described for dna but. Prewarm 50ml of optimum stored in cold room at 4c 10min. Trans it transfection reagents are designed, tested and manufactured in madison, wi.
Fectopro transfection reagent for bioproduction polyplus. You might consider using amaxa technology, according to their protocol you can get 65 % transfection. Mar 27, 2007 too much lipofectamine will be too cytotoxic, and too little nucleic acid will give low efficiency of transfection. Use lipofectamine2000 with plus reagent to transfect your plasmid dna into raw264. Plate an amount that will result in approximately 85 to 90% confluence see table below on the day of transfection. Please refer to table 1 for recommended reaction conditions for other dish or plate sizes.
Transfection guide overview of transfection methods promega. The mechanism of lipofectamine 2000 mediated transmembrane. I have worked with this cell line and i couldnt get a good transfection efficiency with lipofectamine 2000 either. Be sure to mix reagent invert 5 to 10 times prior to taking an aliquot for transfection. Unlike other commercially available reagents, fectopro transfection kit is suitable for both cho and hek293 cells fig. Irf induction in thp1lucia isg cells induction of type i ifns with 23 cgamp can be studied in a variety of cells. Refer to the cell lines database for a list of cell types successfully.
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