Jul 24, 2007 lipofectamine 2000 is a cationic liposome based reagent that provides high transfection efficiency and high levels of transgene expression in a range of mammalian cell types in vitro using a simple protocol. In tube b place 190 l of optimemi and 10 l lipofectamine 2000. This result shows that simple, lipidmediated transfection of hep g2 cells is a viable alternative to gene. In this study, we evaluated the efficiency of two transfection methods, chemically mediated polyethyleneimine pei treatment and physically mediated electroporation, on a rat heart myoblast cell line, h9c2 21. Prepare transfection complexes by mixing 40 l of serum. Optimemin ul, equal volume for dna and lipofectamine lipofectaminein ul 25. 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 transfection of c2c12 cells bridges lab. Invitrogen lipofectamine 3000 transfection reagent fisher. For 96 well plate for me worked well plating approximately cells well transfecting them with 300ng dna and 0.
The intracellular trafficking mechanism of lipofectamine. The reagent provides high transfection efficiency in many cells types and formats. Lipofectamine 2000 reagent protocol 202 lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. Mar 27, 2007 lipofectamine 2000 reagent is a lipidderived reagent designed to deliver exogenic nucleic acids dna andor rna into eukaryotic cells, either adherent or in suspension. Invitrogen lipofectamine 2000 transfection reagent. Researchers use lipofectamine 2000 reagent for sirna and shrnabased gene knockdown experiments, as well as for gene expression studies.
After pei transfection of ppgk1egfp into h9c2 21 cells, egfp expression could be easily detected by fluorospectrometer after 48 h. Cotransfection of two plasmids tissue and cell culture. Lipofectamine is formulated to give high transfection efficiency with little toxicity to cells. Transfection protocol lipofectamine 3000 transfection. Lipofectamine 2000 reagent protocol 202lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. I have been using lipofectamine 2000 with aav293 cells and triple transfection to make aavs. To perform transfection experiments in other cell culture plates, simply multiply the suggested quantities by the relative surface area of your plate.
Lipofectamine 2000 reagent is a lipidderived reagent designed to deliver exogenic nucleic acids dna andor rna into eukaryotic cells, either adherent or in suspension. 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. This protocol is for transfecting plasmid dna into mammalian cells. 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. 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. Plate cells so they will be 7090% confluent at the time of transfection. Invitrogens lipofectamine 2000 is one of many cationic lipidbased transfection reagents presently on the market. Invitrogen lipofectamine 3000 transfection reagent l3000008. Protocol 1 stable transfection using lipofectamine 2000 in 6 well plates. Cells from the h9c2 cell line express ach receptors and are often used as transfection hosts in the study of many myocardial diseases. Hela seed 4 x 105 cells the day before transfection and harvest cells 3648 h posttransfection. In biontex experiments gfp positivity increased to 90.
Lipofectaminez 2000 is a proprietary formulation suitable for the transfection of nucleic acids into eukaryotic cells. Altogen cro offers in vivo rnai services, tumor xenograft models, toxicology testing, stable cell line generation, and. Altogen biosystems provides in vivo transfection reagents, over 100 preoptimized in vitro transfection kits for cell lines and primary cells, and electroporation delivery products. Dnaintm transfection reagent family a comparative study of transfection reagents. Lipofectamine reagents are widely accepted as goldstandard for the safe delivery of exogenous dna or rna into cells. I would like to know if any of you have ever worked with h9c2 cells and successfully transfected them. Lipofectamine 2000 reagent thermo fisher scientific. Lipofectamine 2000 transfection reagent is best transfection reagent available from life technologies for the co transfection of sirna and plasmid dna. Highest transfection efficiency in many cell types and formats e. Mix optimem and lipofectamine in another eppendorf 32 l lipofectamine in 0,8 ml optimem 3. Downregulation of mir3568 protects against ischemia. H9c2 cardiomyocytes lipofectamine 2000 transfection. Prepare plasmid dnalipid complexes recommend 2 doses of lipid.
Despite this, a satisfactory mechanismbased explanation of their. H9c2 cardiomyocytes lipofectamine 2000 transfection conundrum. Lipofectamine 2000 cd reagent complexes can be added directly to cells in culture medium. Transient plasmid dna transfection into hela and hacat. Sep, 2005 transfection of primary synoviocytes resulted in greater than 50% transfection efficiency. Theres a difference between the two protocols lipofectamine 3000 has an extra reagent that needs to be added. Protocol 6 well dish plate cells and grow to 70% confluence in 2 ml media without antibiotics dmem with 10% fbs, no psg. Altogen biosystems produces preoptimized lipidbased highefficiency transfection reagent kits for the h9c2 embryonic rat myocardium cell line. Comparative expression of gfp in h9c2 rat cardiomyocytes transfected with viromer red and lipofectamine 2000 data from v.
Transfection protocol lipofectamine 3000 transfection reagent. Laboratory 2010 lipofectamine 2000 transfection protocol 10cm plates reagents. Several cell densities and lipid volumes were investigated to determine optimal transfection conditions, shown by the shaded boxes. Comparative transfection efficiency of jetprime versus its main competitor.
I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. H9c2 cardiomyocytes were seeded in the sixwell plate for 24 h and that with 50 nm mir3568 mimic sequence. Invitrogen lipofectamine 3000 transfection reagent l3000008 gibco optimem i reduced serum medium 31985062 thermo scientific nunc 24well cellculture treated multidishes 142475 step tube complexation components amount per well 24well. Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. 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. Effi ciency and ofp expression were analyzed 72 hours posttransfection and a u2os and b hepg2.
I use 24well plates for transfections in triplicates. This shows that the lipofectamine 2000 concentration should be optimized for each cell type. Jayarajan, charite berlin, center for cardiovascular research germany % gfp positive cells complete medium serum and antibioticfree medium 14. Invitrogen lipofectamine 3000 transfection reagent. I transfected h9c2 cells with different transfection reagents such as lipofictamine 2000 and 3000, viromer yellow and red. Transfection of nih3t3 cells, hela, swis 3t3, 293t with. Reverse transfection protocol for dna in 96well plates using transitx2 dynamic delivery system. In hacat cells, lipofectamine 2000 achieved a transfection efficiency of 22. Invitrogen claims that this reagent gives the highest protein expression levels in the widest variety of adherent and suspension. Lipofectamine 3000 reagent maintains a high transfection efficiency within a robust dynamic range of lipid doses for quick and easy optimization. Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations.
Lipofectamine 2000 has been used successfully to transfect short interfering rnas sirna into mammalian cells for rna interference rnai studies gitlin et al. This reference provides general guidelines and procedures to transfect sirna into mammalian cells using lipofectamine 2000. Lipofectamine o23, ot, hela, 2a1 wt shsy5y use 10x amount of dna polyfect 292t, hela, mcf7, ot70, otbag references. Mix optimem with dna in an eppendorf 510 g dna0,8 ml optimem 2. If there appears to be largescale loss of cells after lipofectamine treatment, then it might pay to try using polylysinecoated plates. Add 6l of lipofectamine2000 from biostores and 100 l of. The highest transfection efficiency in many cell types and formats e. Transient plasmid dna transfection into hela and hacat cells. Make sufficient 10% fbs in optimem media wo antibiotics sterile filter 2.
Lipofectamine 2000 cd reagent is a proprietary animal originfree formulation for transfecting nucleic acids into eukaryotic cells. Cut plasmid with an appropriate restriction enzyme to produce a linear fragment for transfection. Lipofectamine 2000 transfection reagent from invitrogen. When transfection efficiencies were assayed by flow cytometry after 72 h. The weight ratio of lipofectamine 2000 to pdna was kept constant at 3. Three cell densities of hepg2 cells were transfected with gapd sirna 100 nm using a range of volumes 0. Transfection of mcf7 with lipofectamine 2000 maartje vogel. Invitrogen lipofectamine 2000 transfection reagent fisher.
Transfection reagents are highly efficient for dna and sirna transfection in vivo and in vitro. A highly efficient method of transfecting hep g2 cells. F, and two pulses transfected cells of h9c221 expressed egfp, and peitransfected cells appeared to be less damaged viability 93. Lipofectamine 2000 transfection protocol 10cm plates reagents. May 11, 2016 the intracellular trafficking mechanism of lipofectaminebased transfection reagents and its implication for gene delivery. Lipofectamine 2000 transfection, the expression of gfp was detected in 76,4% of hela cells with a mean fluorescence intensity of 437. Cells should be plated on collagencoated dishes, as for normal propagation. Use a range of several ul of lipofectamine and ug dna to increase viability after transfection and efficiency. Using lipofectaminez 2000 for transfection provides the following advantages. Transfection protocol transfection components component cat. For additional information on scaling your transfection reaction, see page 4. Plate an amount that will result in approximately 85 to 90% confluence see table below on the day of transfection.
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. Exceptional transfection efficiency in the broadest range of cell lines and the highest levels of recombinant protein expression. Transfection was measured by luciferase activity using a conventional assay. Genscript recommends using lipofectamine 2000 for all transfections. 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. A highly efficient method of transfecting hep g2 cells summary. Transfection protocol of lipofectamine rnaimax can be obtained from the invitrogen website. H9c2 7 ipsc colonies 109 ipsc colonies lncap a549 hepg2. Lipofectamine 2000 transfection for hek293 cells about 6070% transfection rate transfect cells 6well plate. 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. 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.
Efficiency of dna transfection of rat heart myoblast cells. Thee two most important factors in choosing transfecting reagent are. 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. Add 100l of optimum to the eppendorf tube to dilute the dna and mix by tapping. Cotransfection of two plasmids need the protocol for lipofectamine 2000 based transfection nov 012009 hi, i am trying to cotransfect transient trasnfection two different plasmids in to hek293t cells.
H9c2 transfection reagent embryonic myocardium altogen. However, for cotransfection of sirnachimera and plasmid dna, lipofectamine 2000 should be used. This result shows that simple, lipidmediated transfection of hep g2 cells is a viable alternative to gene delivery methods such. Superior performance for co transfection of sirna and plasmid dna. The transfection efficiencies of the pte cells were 5% effectene. Optimized conditions for successful transfection of human. The mechanism of lipofectamine 2000 mediated transmembrane. A sample protocol is listed here for transfection experiments performed in 6well plates. Transfection efficiency was assessed by facs analysis in various cell lines 24 h after transfection in 24well plates. In tube a place 2 g of dna in optimemi for a final volume of 200 l. Lipofectamine 2000 transfection reagent is versatile transfection reagent that has been shown to effectively transfect widest variety of adherent and suspension cell lines. Lipofectamine 3000 transfection reagent leverages our most advanced lipid nanoparticle technology to provide superior transfection performance with improved application outcomes and reproducible results. Prewarm 50ml of optimum stored in cold room at 4c 10min. Transfection amounts component 96well 24well 6well dna per well 100 ng 500 ng 2500 ng p3000 reagent per well 0.
988 999 810 702 158 705 1657 379 1294 259 1382 610 74 1021 1618 1014 1276 1410 1277 843 1453 793 1511 558 888 1036 151 811 655 1306 1024 735 1486 141 1032 368 1167 77 1510 1039 45 642 176 1156 1089 804