A549 lipofectamine 3000 lipofectamine 2000 fugene hd lipofectamine 2000 lipofectamine 3000 competitor 1 competitor 2 western blot, hepg2 gststat. How to maximize transfection efficiency in a549 cells. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. Ordering information gibco lung cancer starter kit. Lipofectamine 2000 reagent can be used for transfection of plasmid dna and small rnas. Reverse transfection protocol for dna in 96well plates using transitx2 dynamic delivery system. Table 2 examines transfection efficiency and cell viability under optimal conditions for each reagent 24 hours after transfection. Selected citations for a549 transfection reagent used in the following publications. Lipofectamine 2000 cd reagent complexes can be added directly to cells in culture medium. Assay measure protein expression introduction this protocol shows optimum transfection condition using hilymax in a549 cells. A sample protocol is listed here for transfection experiments performed in 6well plates. Improved delivery of cas9 proteingrna complexes using lipofectamine crisprmax. Maintain p19 cells undifferentiated in mem with 10% serum 7. To transfect cells with sirna, follow the protocol as.
The weight ratio of lipofectamine 2000 to pdna was kept constant at 3. It is possible to plate and transfect the cells on the same day, but cells must adhere to the bottom of the 6well dish. The reagent provides high transfection efficiency in many cells types and formats. Genscript recommends using lipofectamine 2000 for all transfections. Transitx2 for crispr plasmid delivery protocol pdf. Transient plasmid dna transfection into hela and hacat cells. Lipofectaminez 2000 is a proprietary formulation suitable for the transfection of nucleic acids into eukaryotic cells. Find msds or sds, a coa, data sheets and more information. Invitrogen lipofectamine 3000 transfection reagent fisher. The intracellular trafficking mechanism of lipofectamine.
Transfection protocol of lipofectamine rnaimax can be obtained from the invitrogen website. However, for cotransfection of sirnachimera and plasmid dna, lipofectamine 2000 should be used. Lipofectamine 2000 transfection for hek293 cells about 6070% transfection rate transfect cells 6well plate. Gibco rpmi 1640 with glutamax supplement 61870036 10% gibco fbs a3160401 1.
I have been transfecting a549 cells for long time but still could not get transfection efficiency more than 30%. Use a range of several ul of lipofectamine and ug dna to increase viability after transfection and efficiency. The multiple cloning sites mcs sequence in plv cdna vectors. Optimized conditions for successful transfection of human. A549 transfection kit lung carcinoma, ccl185 altogen. Commercially available a549 transfection reagent is provided by altogen biosystems link to manufacturer product page a549 transfection kit. Transfecting a human neuroblastoma cell line with monster. Lipofectamine 2000 transfection reagent is best transfection reagent available from life technologies for the co transfection of sirna and plasmid dna. Feb 18, 2016 to identify the best transfection reagents for delivery of cas9 rnps, initially we compared several commercially available protein transfection reagents, including lipofectamine 2000, lipofectamine 3000, lipofectamine rnaimax, lipofectamine meassengermax, turbofect, and xfect protein transfection reagent. Transient plasmid dna transfection into hela and hacat.
I use 24well plates for transfections in triplicates. Transfection protocol transfection components component cat. Lipofectamine 2000 reagent protocol 202 lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. Scaling up or down lipofectamine 3000 reagent transfections. Have you tried recovery step in your transfection protocol. Plate cells so they will be 7090% confluent at the time of transfection. Singlecell transfection of adherent cells has been accomplished using singlecell electroporation scep with a pulled capillary. Enhanced transfection efficiency using lipofectamine 3000 reagent. The impact of transfection mediated toxicity gene expression and cytotoxicity analysis of transfection reagents introduction while plasmid dna delivery is a widely used method to study cellular functions of proteins of interest, studies to identify nontoxic gene delivery reagents are limited. Introducing the versatile reagent with the power to. A549 cells are an important model for drug metabolism and are also used as a host for gene transfection.
Transitx2 dynamic delivery system and lipofectamine 2000 transfection reagent were used to. Transfection reagent for a549 cells lung carcinoma cells, ccl185 two component formulation enhances lipid mediated transfection efficiency. Transfecting plasmid dna into a549 cells using lipofectamine. Lipofectamine 2000 transfection reagent is versatile transfection reagent that has been shown to effectively transfect widest variety of adherent and suspension cell lines. Transfection protocol of adherent a549 cells 96well plate 1. 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.
Transfection of nih3t3 cells, hela, swis 3t3, 293t with. Two component lipidbased proprietary formulation optimized for a549 cells transfection. Invitrogen lipofectamine 2000 transfection reagent. A549 a or mdck b cells were transfected with luciferase encoding. Make sufficient 10% fbs in optimem media wo antibiotics sterile filter 2. 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. Fugene hd and lipofectamine 2000 should also work as i have seen them. Highest transfection efficiency in many cell types and formats e.
Lipofectamine 2000 cd reagent is a proprietary animal originfree formulation for transfecting nucleic acids into eukaryotic cells. Each cell line was tested for with the neon 24 optimization protocol. The intracellular trafficking mechanism of lipofectamine based transfection reagents and its implication for gene delivery. Singlecell transfection by electroporation using an. Lipofection is a passive method for the introduction of dna into cells, using synthetic cationic lipids that interact with dna to form lipiddna complexes, which fuse with the cell membrane and deliver the dna to the cell. Dna transfection lab word dna transfection of lacz gene. 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. Optimized delivery of sirna into 3d tumor spheroid. Lipofectamine 3000 transfection reagent leverages our most advanced lipid nanoparticle technology to provide superior transfection performance with improved application outcomes and reproducible results. You can calculate this according to instructions in your reagent manual. Hek 293ft, a549 and n2a cells were maintained in dmem medium. This shows that the lipofectamine 2000 concentration should be optimized for each cell type.
Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. Superior performance for co transfection of sirna and plasmid dna. Transfection protocol lipofectamine 3000 transfection reagent a549 lung cancer cells complete growth medium component cat. Even though cell lines differ in their experimental properties, this protocol will yield efficient transfection at low nano molar concentrations in most of the commonly. Laboratory 2010 lipofectamine 2000 transfection protocol 10cm plates reagents. Lipofectamine 2000 reagent thermo fisher scientific. A kozak consensus sequence gccacc before start codon is highly recommended. Optimized easytouse transfection protocol provided for transfection of sirna, dna, mrna, and microrna.
Transfecting plasmid dna into a549 cells using lipofectamine 3000 reagent. To perform transfection experiments in other cell culture plates, simply multiply the suggested quantities by the relative surface area of your plate. Transfection guide overview of transfection methods promega. Improved delivery of cas9 proteingrna complexes using. A549 cell optimum transfection condition optimum condition for transfection for 24well plate table 1 optimum condition for tranfection to a549 cells cell density serumfree medium 30 l dna 1 g hilymax 4.
Viafect reagent offers performance similar to or better than that of lipofectamine 2000 reagent, and it may be suitable. Cy3 sirna formed in optimem using lipofectamine 2000 at 2, 6 and 24 hours post transfection. Higher densities can lead to spontaneous differentiation. Several cell densities and lipid volumes were investigated to determine optimal transfection conditions, shown by the shaded boxes. Cas9 rnps delivered into u2os cells using lipofectamine 2000 resulted in up to 80 % modification efficiency at an integrated gfp reporter and cas9 rnps. Each reagent was used to transfect hek 293, hela, lncap, a549, and hepg2 cells in a 96well format, and. Sep, 2005 transfection of primary synoviocytes resulted in greater than 50% transfection efficiency. Lipofectamine 2000 reagent protocol 202lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. A dynamic transfection system for the delivery of plasmid dna, sirnamirna and. Mix optimem with dna in an eppendorf 510 g dna0,8 ml optimem 2. To transfect cells with sirna, follow the protocol as described for dna but. Introducing the monster green fluorescent protein phmgfp vector using the transfast transfection reagent and the optimized transfection conditions resulted in the cell notes issue 7 2003 14 neuroscience transfecting a human neuroblastoma cell line with monster green fluorescent protein by natalie betz, ph. A549 transfection reagent is supplied in liquid form at a concentration of 0.
Transfection protocol lipofectamine 3000 transfection. Exceptional transfection efficiency in the broadest range of cell lines and the highest levels of recombinant protein expression. Version 1, 41 5 g pipet up and down 1215 times with a 10 ml pipet until a single cell suspension is obtained. Scaling up or down lipofectamine 3000 reagent transfections use the following table to scale the volumes for your transfection experiment. Introduction lipofectamine ltx reagent is a proprietary, animalorigin free formulation for the transfection of dna into eukaryotic cells with low cytotoxicity. Change the growth medium 4 hours after transfection. This protocol is for transfecting plasmid dna into mammalian cells. Different cell lines were transfected with a gfp reporter vector using the indicated transfection reagents. Transfection protocol lipofectamine 3000 transfection reagent. Transfection protocol of adherent a549 cells 96well plate. Add 100l of optimum to the eppendorf tube to dilute the dna and mix by tapping.
Transfection p19 cells with lipofectamine 2000 the same protocol can be used for 293t cells 1. Prepare plasmid dnalipid complexes recommend 2 doses of lipid. Cut plasmid with an appropriate restriction enzyme to produce a linear fragment for transfection. For additional information on scaling your transfection reaction, see page 4. Reverse transfection is the transfer of genetic material into cells and is reverse because the order of dna and cells is reverse that of conventional transfection. However, significant toxicity from lipofectamine 2000 was also reported in u2os cells. Invitrogen lipofectamine 3000 transfection reagent 1. It is possible to plate and transfect the cells on the same day, but cells must adhere to. At 72 hours posttransfection the cells were analyzed by western blot for protein expression levels normalized by total protein, 10 g of total protein loaded per each well. We have also used lipofectamine 2000 to introduce sirna into primary synoviocytes and obtained a comparable transfection efficiency when transfecting larger dna species scott et. A reverse transfection protocol was used to transfect mcf7 and hepg2 cells. Prewarm 50ml of optimum stored in cold room at 4c 10min.
Mix optimem and lipofectamine in another eppendorf 32 l lipofectamine in 0,8 ml optimem 3. 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. Optimum amount needed is determined from the protocol for lipofectamine 3000 transfection reagent. Three cell densities of hepg2 cells were transfected with gapd sirna 100 nm using a range of volumes 0. Using lipofectaminez 2000 for transfection provides the following advantages. Choosing the right transfection reagent for optimal efficiency.
Lipofectamine 2000 transfection protocol 10cm plates reagents. Hepesbuffered physiological saline solution containing pegfp plasmid at a low concentration 0. Thermo scientific nunc 24well cellculture treated multidishes 142475 invitrogen lipofectamine 3000 transfection reagent l3000008 gibco optimem i reduced serum medium 31985062 step tube complexation components amount per well 24well. May 15, 2009 singlecell transfection of adherent cells has been accomplished using singlecell electroporation scep with a pulled capillary. 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. This protocol isintended for the transfection of adherent mammalian tissue culture cells. The mechanism of lipofectamine 2000 mediated transmembrane. The transfection reagent lipofectamine 2000 lf2000, invitrogen is preferred for transfection. Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations.
This reference provides a recommended procedure to transfect plasmid dna into human a549 lung carcinoma cells atcc, cat. Achieve superior transfection efficiency while maintaining low toxicity lipofectamine 3000 reagent maintains a high transfection efficiency within a robust dynamic range of lipid doses for quick and easy optimization. Add 6l of lipofectamine2000 from biostores and 100 l of. Protocol 1 stable transfection using lipofectamine 2000 in 6 well plates. May 11, 2016 the intracellular trafficking mechanism of lipofectaminebased transfection reagents and its implication for gene delivery. A549 transfection reagent is tested functionally by transfection of a549 cells with a small interfering.
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