Sep 15, 2017 for transfection, a549 cells were seeded with a density of 150,000 cellswell into a 12well plate. Transfection protocol lipofectamine 3000 transfection. Start any new transfection by testing the recommended four. Dna lipofectamine 2000 cd complexes can be added directly to cells in culture medium. Coronary artery coa and aortic ao smc and ec were transfected with a reporter plasmid, encoding chloramphenicol acetyltransferase type 1 cat, with seven different transfection reagents, two electroporation methods and a photochemical. Does lipofectamine 2000 give a stable transfection. Human mesenchymal stem cells lipofectamine 2000 thermo. Myostatin is a growthpromoting factor that inhibits muscle growth during the myogenic process.
Reliable and e cient for workhorse and easytotransfect cell lines with a simple onetube protocol, it has been trusted by scientists since 1999. Application note gene delivery solutions stem cell. Test serumfree media for compatibility with lipofectamine. Lipofectamine 3000 transfection kit thermo fisher bioz. Make sure you remove any antibiotics from the media 24 hours before transfection and the cells need. Transfection efficiencies lower than 50% were not reported in this table. A549 a or mdck b cells were transfected with luciferase encoding plasmid dna using either transitx2 mirus bio, lipofectamine 2000 thermo fisher scientific or lipofectamine 3000 thermo fisher scientific for 24 hours at indicated reagenttodna ratios or reagenttop3000todna ratio. Lipofectamine 2000 transfection reagent dna transfection. The animal originfree formulation ensures that mammalian cell culture and bioproduction processes are free of animalderived materials.
The weight ratio of lipofectamine 2000 to pdna was kept constant at 3. The amount of lipofectamine 3000 reagent for successful transfection varies. Cells were transfected with a firefly luciferase encoding plasmid dna 0. Invitrogen lipofectamine 2000 transfection reagent life. Apr 18, 2005 the aim of this study was to determine the optimal nonviral transfection method for use in human smooth muscle cells smc and endothelial cells ec.
I am using l929 cells atcc ccl1 for transfection with lipofectamine 2000. Lipofectamine 2000 reagent thermo fisher scientific. Lipofectamine 2000 reagent is a proprietary formulation that facilitates highly efficient delivery of stealth rnai or short interfering rna sirna to mammalian cells for rnai analysis. Lipofectamine 2000 is a really good transfection reagent that can warranty you a high transfection efficiency. Cells in a 6 well dish, plated and at 70% confluence. Metafectene pro had less toxicity than lipofectamine. Mammalian cell transfection can be done utilizing a variety of techniques. Comparison of transfection efficiency between commercialy. Visit our product page for additional information and protocols. The trusted, simple, broadspectrum transfection reagent for most cell lines. Scaling up or down lipofectamine 3000 reagent transfections. Lipofectamine 3000 reagent thermo fisher scientific us. Viafect transfection reagent transfection assay e4981.
Researchers use lipofectamine 2000 reagent for sirna and shrnabased gene knockdown experiments, as well as for gene expression studies. 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. Increasing the post transfection period increases toxicity. Lipofectamine 2000 cd reagent is a proprietary animal originfree formulation for transfecting. Lipofectamine 2000 cd reagent complexes can be added directly to cells in culture medium. Comparative transfection efficiency of jetprime versus its main competitor. Genscript recommends using lipofectamine 2000 for all transfections. Lipofectamine 2000 transfection reagent is versatile transfection reagent that has been shown to effectively transfect widest variety of adherent and suspension cell lines. Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations. Transfection amounts component 96well 24well 6well dna per well 100 ng 500 ng 2500 ng plus reagent per well 0. Lipofectamine 2000 transfection reagent is a superior choice for cotransfection, given its effectiveness for transfecting both sirna and plasmid dna ideally suited for highthroughput work because transfection conditions can be easily established for automated or robotic systems a highperformance workhorse transfection reagent. I am trying to optimize the transfection by using lipofectamine 2000 for 2000 ng of plasmid dna well with different amounts of lipofectamine 2000 5ul, 10ul in the 6well plate. The reagent provides high transfection efficiency in many cells types and formats. Lipofectamine 2000 transfection reagent is a proprietary formulation for transfecting.
A sample protocol is listed here for transfection experiments performed in 6well plates. Transitx2 dynamic delivery system outperforms lipofectamine reagents. Gibco hams f12 nutrient mix 11765054 5% horse serum 1 mm gibco sodium pyruvate 160070 25 mm gibco hepes 15630106 proper culture techniques and procedures are an essential. The intracellular trafficking mechanism of lipofectamine. Optimizing transfection lipofectamine 2000 cd reagent. High transfection effi ciency enables lipofectamine 3000 to be used for a variety of applications, including reprogramming of fi broblasts, gene editing, and transfection of diffi cult cells. It is not necessary to remove complexes or changeadd medium after transfection, but complexes may be removed after 46 hours. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. Lipofectamine 3000 is the best choice for transfection of chok1 and hek293 with pcdh while turbofect is preferably used in transfecting these cell lines with pegfpn1 tab. Does anyone knows an efficient protocol to transfect human.
How to increase the transfection efficiency in primary. Manual muscle testing was developed for the first time in the world during the 18601880 century by swedish physiotherapists from the royal central institute of. Lipofectamine 2000 is tested for absence of microbial contamination with blood agar plates, sabaraud dextrose agar plates, and fluid thioglycolate medium, and functionally by transfection of chok1 cells with a reporter plasmid. Transfection guide overview of transfection methods promega. Expressing, mutagenesis, transfection, incubation journal.
Protocol for human skeletal muscle myoblasts hsmm transfection by fugene hd 1. Intube transfection improves the efficiency of gene transfer in. Adherent monolayer cultures were transfected using lipofectamine 2000 0. Their transfection performances were compared to those of lipofectamine 2000 and lipofectamine 3000 lf3k. Rapid and highly efficient mammalian cell engineering via. Sep 19, 2018 luciferase activity was measured post transfection of a luciferase coding plasmid using different chemical transfection reagents, transitx2, lipofectamine 2000, and lipofectamine 3000. Stealthsirna transfection protocol lipofectamine 2000.
Lipofectamine 2000 cd chemically defined transfection reagent is a proprietary animal originfree formulation for transfecting nucleic acids into eukaryotic. Thermo fisher u251 cells lipofectamine 2000 lipofectamine 2000 transfection reagent is a versatile transfection reagent that has been shown to effectively transfect the widest variety of adherent and suspension cell lines researchers use lipofectamine 2000 reagent for sirna and shrna based gene knockdown experiments as well as for gene expression studies with lipofectamine 2000 transfection. Today i made a test, i transfected myoblast with gfp in following conditions. Conditions were used according to the manufacturers recommendation for both lipofectamine 2000 and for jetprime. Lipofectmax transfection reagent has been tested to work the same efficiency as lipofectamine 2000 reagent, and used for the transfection of both. See how well this product performs for dna transfection mammalian cells primary cells human pulmonary artery smooth muscle cells hpasmc. Dnalipofectamine 2000 cd complexes can be added directly to cells in culture medium it is not necessary to remove complexes or changeadd medium after transfection, but complexes may be removed after 46 hours pcr and realtime pcr, reverse transcription, cdna synthesis direct from cells. Cadherin11 regulates both mesenchymal stem cell differentiation into smooth muscle cells and the development of. Aug 20, 2015 one day prior to transfection, the cells were seeded in a 24well plate at a cell density of 12. Here we compared the transfection and viral transduction efficiency using commercially available transfection reagents including fugene 6, lipofectamine 2000 and lipofectamine 3000 in different cell lines and primary cultured cells.
When a finite cell line undergoes transformation and acquires the ability to. To date, how to reach the maximum transfection and viral transduction efficiency is still challenging. Comparison of transfection efficiency of polymerbased and. Transfection reagents and sso rnaimax transfection reagent 778030, invitrogen, waltham, ma, usa, lipofectamine 3000 l3000001, invitrogen, fugene transfection reagent e2311, promega, sydney, australia, lipofectamine 2000 reagent 11668027, invitrogen, lipofectin reagent 18292011, invitrogen were used in this study. Viafect reagent offers performance similar to or better than that of lipofectamine 2000 reagent, and it may be suitable for some cell lines where other lipidbased reagents result in lower transfection efficiencies table 1. For hek293t cells we find that 1520 minutes is optimal. Lipofectamine 2000 cd reagent is a proprietary animal originfree formulation for transfecting nucleic acids into eukaryotic cells. Lipofectamine transfection reagents shipped at ambient. Is lipofectamine 2000 the optimum reagent for tranfection. Lipofectamine 3000 transfection reagent the preferred choice for transfection of two types of stem cells. Lipofectaminez 2000 is a proprietary formulation suitable for the transfection of nucleic acids into eukaryotic cells. Transfection commonly refers to the introduction of nucleic acids into eukaryotic cells, or more specifically, into animal cells. I want to try to use hela cell and lipofectamine 2000 to. Invitrogen lipofectamine 2000 cd transfection reagent.
Invitrogen lipofectamine 2000 transfection reagent is a versatile transfection reagent that has been shown to effectively transfect a variety of payloads into the widest variety of adherent. Several cell and application specific dnain transfection reagents also have been developed. To perform transfection experiments in other cell culture plates, simply multiply the suggested quantities by the relative surface area of your plate. Is transfection efficiency of c2c12 cells with lipofectamine. Lipofectamine 2000 thermo fisher, xtremegene roche, hiperfect qiagen, and transittko mirus reagents. Start any new transfection by testing the recommended two concentrations of lipofectamine 3000 reagent to determine an optimum amount. The mechanism of lipofectamine 2000 mediated transmembrane. Each reagent was used to transfect hek 293, hela, lncap, a549, and hepg2 cells in a 96well format, and. Lipofectamine 2000 is a proprietary formulation for the transfection of nucleic acids. For researchers interested in other delivery methods, we also offer additional invitrogen lipofectamine reagents for mrna transfection and lentiviral production as well as the invitrogen neon transfection system for.
Lipofectamine 3000 reagent thermo fisher scientific. For additional information on scaling your transfection reaction, see page 4. Lipofectamine 3000 reagent for pluripotent stem cell applications. For the following protocol, we specify volumes for a 12well plate. The highest transfection efficiency in many cell types and formats e. Lipofectamine 2000, supplied by thermo fisher, used in various techniques. D00120620 1 day after induction of mutant htt expression lipofectamine 2000, invitrogen. I use 24well plates for transfections in triplicates.
To obtain the highest transfection efficiency and low nonspecific effects, optimize transfection conditions by varying rna and lipofectamine 2000 concentrations. Lipofectamine 2000 cd reagent thermo fisher scientific. When i use lipofectamine 2000 to transfect huvecs primary endothelial cells, i have to. Lipofectamine messengermax reagent protocol outline a. Plate cells so they will be 7090% confluent at the time of transfection.
Transfection guide overview of transfection methods. Optimizing cardiomyocyte transfection with lipofectamine. Incubate cells at 37c in a co2 incubator for 1848 hours prior to te. Highest transfection efficiency in many cell types and formats. Hepg2 with up to 50% transfection efficiency and excellent cell viability. Prior to use of lipofection on an alternative cell line, we strongly recommend doing. We utilize lipofection lipofectamine 2000 for transient gene expression. Transfection amounts component 96well 24well 6well final mrna per well 100 ng 500 ng 2500 ng final lipofectamine. The transfection mix consisted of mrna, lipofectamine 2000 l2k and optimem both. The stability of the transfection is mainly dependent on the plasmid. Lipofectamine 2000 reagent protocol 202 lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. Transfection efficiency was assessed by facs analysis in various cell lines 24 h after transfection in 24well plates. Lipofectamine transfection of c2c12 cells bridges lab.
There are dozens of transfection reagents commercially available with varying properties and a. We dissect at p0, plate in neurobasal a with b27, glutamax and penstrep transfections are at 610 div, transfection medium is on neurons for 3 hours, both 0. I have used lipofectamine 2000 with h9c2s with a ratio of 6. A simple, highly efficient method for heterologous expression in.
H9c2 cardiomyocytes lipofectamine 2000 transfection conundrum. Lipofectamine 2000 transfection reagent 11668027 0. Electroporation by nucleofector is the best nonviral. The vector contained an ofp reporter gene and was transfected. The neurons were identified and counted manually from the phase contrast image. Protocol for human skeletal muscle myoblasts transfection. 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.
Transfection optimizationhow to get it right biocompare. When myostatin is active, cells quickly differentiate into muscle cells that prevent their growth. Promofectinhepatocyte has been designed for transfection of cells expressing galactosespecific membrane lectins and has been very successfully used to transfect primary hepatocytes as well as hepatocyte cell lines e. Transfection protocol lipofectamine 3000 transfection reagent. Other cell lines may be refractory to some or most of these reagents. Using lipofectaminez 2000 for transfection provides the following advantages. The transfection protocol for lipofectamine 3000 was developed to be easy to use while still ensuring optimum performance and. Lipofectamine 3000 reagent provided higher gfp transfection efficiency than lipofectamine 2000 and fugene hd reagents for all five cell lines. Invitrogen lipofectamine 2000 transfection reagent is the mostcited lipidbased reagent in scienti.
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