Lipofectamine 2000 reagent delivers dna or sirna with excellent transfection performance for protein expression, gene silencing, and functional assays. However, for cotransfection of sirnachimera and plasmid dna, lipofectamine 2000 should be used. Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. Transfection and genome engineering fisher scientific.
Lipofectamine 2000 dna transfection reagent protocol. Superior performance for co transfection of sirna and plasmid dna with reliable performance for highthroughput applications. For successful transfection of gfp plasmid dna into chok1 cells, the optimal dna. A polymerbased transfection caco2 kit for colon adenocarcinoma cells yields at least 94% transfection efficiency and is a helpful tool for gene expression studies as well as for assessing cellular functionality. For additional information on scaling your transfection reaction, see page 4. Genscript recommends using lipofectamine 2000 for all transfections. Exceptional transfection efficiency in a broad range of cell lines with high levels of recombinant protein expression and a simple protocol. Lipofectamine contains lipid subunits that can form liposomes in an aqueous environment. Highest transfection efficiency in cells at 90% confluency.
Evaluation of transfection methods for transient gene. In this study, we evaluated the transfection and viral transduction efficiency using commercially available transfection reagents including fu gene 6 transfection reagent, lipofectamine 2000 and lipofectamine 3000 reagents in dif. 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. 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 breast cancer. Shear stress increases cytotoxicity and reduces transfection. Introducing the versatile reagent with the power to transfect your. The mechanism of lipofectamine 2000 mediated transmembrane. Transfection efficiency and protein expression using geneart crispr nuclease vector. Highest transfection efficiency in many cell types and formats e. The invitrogen lipofectamine 3000 transfection reagent leverages our most advanced lipid nanoparticle technology to enable superior transfection. 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.
Start any new transfection by testing the recommended four concentrations of lipofectamine 2000 reagent to determine an optimum amount. It is best to use polypropylene instead of polystyrene tubes. Original article an improved method for increasing the. Add 6l of lipofectamine2000 from biostores and 100 l of. Lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart.
Bernhard neuhaus a, benjamin tosun a, olga rotan a, annika frede b, astrid m. Superior performance for co transfection of sirna and plasmid dna. Lipofectamine 2000, 3000, messengermax, and rnaimax. To optimize the amount of lipofectamine 2000 for transfection in a 24well plate, start. Lipofectamine 2000 transfection reagent is best transfection reagent available from life technologies for the cotransfection of sirna and plasmid dna.
Flick each 50 ml conical tube and let sit for 5 minutes at room temperature. Plate cells so they will be 6080% confluent at the time of transfection. Transfection protocol lipofectamine 3000 transfection reagent. We did not find substantial transfection efficiency variations in different batches of lipofectamine 2000. The animal originfree formulation ensures that mammalian cell culture and bioproduction processes are free of animalderived materials. Label each tube with a different viral factor oct4, sox2, klf4, or cmyc. The amount of lipofectamine 2000 reagent required for successful transfection varies depending on the cell type and passage number. Dna and different lipofectamine 2000 volumes on cell transfection efficiency was evaluated.
A sample protocol is listed here for transfection experiments performed in 6well plates. Does lipofectamine 2000 give a stable transfection. The i nhibitors of endocytosis were used to treat hep2 cells before lipofectamine 2000pgfpn2 transfection. Lipofectamine 2000 is a very efficient transfection agent, the stability of your knockdown or your overexpression is only related to the half life of your sirna, plamid in the cell. Lipofectamine 2000 cd reagent complexes can be added directly to cells in culture medium. Optimized conditions for successful transfection of human. Product isguaranteed for 6 months from the date of shipment if stored properly. Im trying to express my proteins in nih3t3s, which ive heard are supposedly easy to transfect. Ive heard people claim some very high transfection efficiencies using lipofectamine 2000 or brand x lipid reagent.
Lipofectamine 2000 reagent thermo fisher scientific. The transfection efficacy of lipofectamine 2000 was compromised by its high toxicity, which may adversely affect its application in most cells. The highest transfection efficiencies obtained in both cells are similar to the positive control, lipofectamine 2000. A highly efficient method of transfecting hep g2 cells summary. Lipofectamine 2000 cd reagent complexes can be added. Lipofectamine 2000 cd reagent is a proprietary animal originfree formulation for transfecting nucleic acids into eukaryotic cells. Lipofectamine 3000 transfection reagent hepg2 liver. Exceptional transfection efficiency in the broadest range of cell lines and the highest levels of recombinant protein expression.
May 11, 2016 the intracellular trafficking mechanism of lipofectaminebased transfection reagents and its implication for gene delivery. Pdf optimization of sw480 colon cancer cells transfection with. This study shows that lipofectamine 2000 is an efficient reagent for the delivery of genes into sw480 cells. At the foundation of the popularity of this broadspectrum reagent is the reliably high efficiency and simple protocol for success across a broad range of cell lines figure 1. High transfection efficiencies and high protein expression levels.
Transfection efficiency transfection efficiency of lipofectamine 2000, measured as expression of gfp, was determined against a titration of the reagent in multiple cell lines as. For gene silencing, lipofectamine 2000 transfection reagents highefficiency transfections provide the high levels of gene knockdown needed to achieve convincing results. Lipofectamine 2000 transfection reagent dartmouth college. Metafectene pro had less toxicity than lipofectamine. 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.
Metafectene pro and lipofectamine had the highest efficiencies. Lipofectamine 2000 transfection reagent is the number one choice for cotransfection, given its effectiveness for transfecting both sirna and plasmid dna. Optimizing transfection lipofectamine 2000 cd reagent. Transitpro employs both mecha nisms of action and works by forming lipopolyplexes. This study compared six commercially available reagents arrestin, expressfect, fugene hd, jetpei, lipofectamine 2000, and superfect for gene transfection. Our results show decreased cell growth and transfection efficiency upon. The intracellular trafficking mechanism of lipofectamine. The lipofectamine rnaimax reagent is designed specifically for the delivery of sirna and mirna, and lipofectamine messengermax reagent delivers mrna. Transfection efficiencies were optimal around 40% of transfected cells per spot with 11. Refer to the cell lines database for a list of cell types successfully transfected. Superior performance for cotransfection of sirna and plasmid dna. Lipidmediated dna and sirna transfection efficiency. Lipofectamine rnaimax transfection protocol see page 2 to view a typical rnaimax transfection procedure. Use a range of several ul of lipofectamine and ug dna.
On the day of transfection, which should be 1 day following cell plating, perform the following steps, which have been optimized for a single well of a 24well plate using lipofectamine 3000 transfection reagent. To perform transfection experiments in other cell culture plates, simply multiply the suggested quantities by the relative surface area of your plate. What is a typical transfection efficiency in % for any of you using lipid based reagents and what cell lines did you use. Transfection efficiency comparison in hela cells using transfectamine. Lipofectamine 2000 transfection for hek293 cells about 6070% transfection rate transfect cells 6well plate. A b on the day of transfection, which should be 1 day following cell plating, perform the following steps, which have been optimized for a single well of a 24well plate using invitrogen lipofectamine 3000 transfection reagent. Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations. Lipofectamine 2000 had highest efficiency but also highest toxic effect.
Altogen biosystems provides efficient biodegradable transfection reagent kits for the caco2 colon cancer cell line. Highest transfection efficiency in many cell types and formats. The i nhibitors of endocytosis were used to treat hep2 cells before lipofectamine 2000 pgfpn2 transfection. Invitrogen lipofectamine 2000 transfection reagent. The intracellular trafficking mechanism of lipofectaminebased transfection reagents and its implication for gene delivery. Prewarm 50ml of optimum stored in cold room at 4c 10min. Lipofectmax transfection reagent is a lipidbased transfection reagent that forms a complex with dna or rna, and transports the complex into a variety of adherent and suspension cell lines. Assay of transfection efficiency on the 2100 bioanalyzer in this experiment, pegfpc2. Transfection protocol of lipofectamine rnaimax can be obtained from the invitrogen website. In this study, we evaluated the transfection and viral transduction efficiency using commercially available transfection reagents including fu gene 6 transfection reagent, lipofectamine 2000 and. Invitrogen lipofectamine 2000 transfection reagent 0. Overwhelmingly more publications cite the use of lipofectamine.
Comparison of transfection efficiency of nonviral gene. Add 100l of optimum to the eppendorf tube to dilute the dna and mix by tapping. The reagent provides high transfection efficiency in many cells types and formats. A highly efficient method of transfecting hep g2 cells. Lipofectamine 2000 reagent protocol 202lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. Each tube gets 8mls of optimem, 32ug of gag pol, 12. Comparison of transfection efficiency between commercialy. 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. Lipofectamine or lipofectamine 2000 is a common transfection reagent, produced and sold by invitrogen, used in molecular and cellular biology. Westendorf b and matthias epple a a inorganic chemistry and center for nanointegration duisburgessen cenide, university of duisburgessen, universitaetsstr.
Transfection efficiency was evaluated with green fluorescence protein gfp expression assays. Pdf optimization of sw480 colon cancer cells transfection. 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 breast cancer cells, hela human cervical cancer. Lipofectamine transfection reagent is a trusted, value reagent with many years of cited use in thousands of publications and with many cell lines. Descriptionlipofectamine 2000 is a proprietary formulation suitable for the. Superior performance for cotransfection of sirna and plasmid dna with reliable performance for highthroughput applications. From successful transfections, we await in our laboratory high transfection efficiency and low rates of apoptosis.
Transfection efficiency of p19 cells is between 3050% from 5099% for many other stable cell lines. Much better transfection efficiency with all reagents. May 05, 20 in general, lipopeptidedna molar ratios of 8. For hela cells we have already achieved a transfection efficiency of 87, 8 97,8 % with lipofectamine. Nucleic acid lipofectamine 2000 complexes can be added directly to cells in culture medium, in the presence or absence of serum. Lipofectamine 2000 transfection reagent is best transfection reagent available from life technologies for the co transfection of sirna and plasmid dna.
Clathrin and caveolaemediated endocytic pathway contributions to transfection efficiency were studied. Although lipofectamine 2000 is compatible with most cell lines, the transfection efficiency cannot be sustained in prolonged high cell density culture for large scale operation 1. Lipofectamine transfection reagents shipped at ambient. Lipofectamine 2000 cd reagent thermo fisher scientific. Overwhelmingly more publications cite the use of lipofectamine 2000 transfection. I use 24well plates for transfections in triplicates. Transfection of nih3t3 cells, hela, swis 3t3, 293t with.
Jun 29, 2010 this study compared six commercially available reagents arrestin, expressfect, fugene hd, jetpei, lipofectamine 2000, and superfect for gene transfection. Lipofectamine 2000 and lipofectamine 3000 reagents deliver dna or cotransfect with excellent transfection performance for protein expression, gene silencing, and functional assays. Mammalian cells transfection protocoljavier calvo follow invitrogen lipofectamine 2000 protocol. Lipofectamine transfection reagent is one of our firstgeneration, cationiclipid transfection reagents formulated for the transfection of dna into eukaryotic cells. But unfortunately the survival rate of cells has only been 46,9 61,9 %. It is used to increase the transfection efficiency of rna including mrna and sirna or plasmid dna into in vitro cell cultures by lipofection. Imlf can be transfected using lipofectamine 3000 thermo fisher scientific, waltham, ma, usa but transfection efficiency is higher using recombinant adenovirus described later, which gives very good expression in a shorttime relative to other methods. Lipidmediated dna and sirna transfection efficiency depends. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. This result shows that simple, lipidmediated transfection of hep g2 cells is a viable alternative to gene. Transfection for chos was performed using lipofectamine 2000.
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