In tube a place 2 g of dna in optimemi for a final volume of 200 l. While lipofectamine 2000 reagent demonstrated transfection efficiencies only slightly lower those. It is used to increase the transfection efficiency of rna including mrna and sirna or plasmid dna into in vitro cell cultures by lipofection. H9c2 cardiomyocytes lipofectamine 2000 transfection. H9c2 cardiomyocytes lipofectamine 2000 transfection conundrum. Lipofectamine 3000 transfection reagent leverages our most advanced lipid nanoparticle technology to provide superior transfection performance with improved application outcomes and reproducible results. H9c2 cells were seeded in 6well plates 24h before transfection, incubated at 37. Lipofectamine or lipofectamine 2000 is a common transfection reagent, produced and sold by invitrogen, used in molecular and cellular biology.
Transfection protocol of lipofectamine rnaimax can be obtained from the invitrogen website. Invitrogen lipofectamine 2000 transfection reagent fisher. Objectives to compare the transfection efficiency of three adrb1 rna interference plasmid paavzsgreenadrb1shrna transfected to h9c2 myocardial cell mediated by lipofectamine 2000. H9c2 cardiomyocytes were seeded in the sixwell plate for 24 h and that with 50 nm mir3568 mimic sequence. Gw24e3121 transfecting paavzsgreenadrb1shrna plasmid to. Lipofectamine transfection of c2c12 cells bridges lab.
I transfected h9c2 cells with different transfection reagents such as lipofictamine 2000 and 3000. Viafect transfection reagent transfection assay e4981. Dmem may be used to dilute lipofectaminez 2000, but transfection efficiency may be compromised. 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. The impact of transfection mediated toxicity mirus bio. When the cell density reaches 9095%, transfecting three different target sequences plasmid paavzsgreenadrb11shrna, paavzsgreen. Transfection of nih3t3 cells, hela, swis 3t3, 293t with. Altogen cro offers in vivo rnai services, tumor xenograft models, toxicology testing, stable cell line generation, and. Each cell line was transfected with a luciferase reporter plasmid using either viafect transfection reagent, lipofectamine 2000 transfection reagent or lipofectamine 3000 transfection reagent following manufacturer instructions, applying either 5l 50ng of dna or 10l 100ng of dna of the transfection complex in each well of a 96.
I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. According to our previous study, sirnas transfection was operated using lipofectamine 2000 invitrogen. Reverse transfection protocol for dna in 96well plates using transitx2 dynamic delivery system. Lipofectamine 2000 transfection for hek293 cells about 6070% transfection rate transfect cells 6well plate. Higher densities can lead to spontaneous differentiation. Lipofectamine 2000 transfection reagent from invitrogen. Assa329 optimization of transfection conditions for transfecting paavireshrgfp plasmid to h9c2 cell mediated by lipofectamine 2000 liu xiaoli 1, 2, hong yuan 1, 3. When h9c2 cells reached 85% confluence, plasmids were mixed with lipofectamine 2000. I transfected h9c2 cells with different transfection reagents such as. However, for cotransfection of sirnachimera and plasmid dna, lipofectamine 2000 should be used.
Transient plasmid dna transfection into hela and hacat cells. I would like to know if any of you have ever worked with h9c2 cells and successfully transfected them. Add 6l of lipofectamine2000 from biostores and 100 l of. Efficiency of dna transfection of rat heart myoblast cells h9c221 by either polyethyleneimine or electroporation. For additional information on scaling your transfection reaction, see page 4. Tm328 fugene hd transfection reagent technical manual. Theres a difference between the two protocols lipofectamine 3000 has an extra reagent that needs to be added. Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations. Downregulation of mir3568 protects against ischemia. Prewarm 50ml of optimum stored in cold room at 4c 10min. 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. Viafect reagent offers performance similar to or better than that of lipofectamine 2000 reagent, and it may be suitable for some cell lines.
Plate cells so they will be 7090% confluent at the time of transfection. Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. The transfection protocol for lipofectamine 3000 was developed to be easy to. Does anyone have an optimized protocol for h9c2 transfections. Altogen biosystems produces preoptimized lipidbased highefficiency transfection reagent kits for the h9c2 embryonic rat myocardium cell line. Genscript recommends using lipofectamine 2000 for all transfections. The transfection protocol for lipofectamine 3000 was developed to be easy to use while still ensuring optimum performance and reliability in a wide panel of cell lines. Lipofectamine transfection of c2c12 cells bridges lab protocols.
Jayarajan, charite berlin, center for cardiovascular research germany % gfp positive cells complete medium serum and antibioticfree medium 14. Three cell densities of hepg2 cells were transfected with gapd sirna 100 nm using a range of volumes 0. Invitrogen lipofectamine 3000 transfection reagent. The mechanism of lipofectamine 2000 mediated transmembrane. Transfection protocol transfection components component cat. Lipofectamine 2000 transfection reagent is best transfection reagent available from life technologies for the cotransfection of sirna and plasmid dna. Lipofectamine 3000 reagent thermo fisher scientific kr. Protocol 1 stable transfection using lipofectamine 2000 in 6 well plates. The intracellular trafficking mechanism of lipofectaminebased transfection reagents and its implication for gene delivery. Increased connexin 43 expression improves the migratory and. A sample protocol is listed here for transfection experiments performed in 6well plates. 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.
According to our previous study, sirnas transfection was operated using lipofectamine 2000 invitrogen, usa. Plate an amount that will result in approximately 85 to 90% confluence see table below on the day of transfection. Assa329 optimization of transfection conditions for. Gw24e3121 transfecting paavzsgreenadrb1shrna plasmid. Highest transfection efficiency in many cell types and formats e. I transfected h9c2 cells with different transfection reagents such as lipofictamine 2000 and 3000, viromer yellow and red.
Cells in a 6 well dish, plated and at 70% confluence. Highest transfection efficiency in cells at 90% confluency. 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. Exceptional transfection efficiency in the broadest range of cell lines and the highest levels of recombinant protein expression. Phosphorylated eef2 is sumoylated and induces cardiomyocyte.
Bcrystallin sirna or control scramble sirna, also using lipofectamine 2000 invitrogen. 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. Transfection reagents are highly efficient for dna and sirna transfection in vivo and in vitro. Transfection p19 cells with lipofectamine 2000 the same protocol can be used for 293t cells 1. Altogen biosystems provides in vivo transfection reagents, over 100 preoptimized in vitro transfection kits for cell lines and primary cells, and electroporation delivery products. The weight ratio of lipofectamine 2000 to pdna was kept constant at 3. At 24 h prior to transfection, cells were passed in 100mm plates sarstedt, montreal, canada and as such that they would be approximately 35% confluent at the time of transfection.
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 thermo fisher scientific. If there appears to be largescale loss of cells after lipofectamine treatment, then it might pay to try using polylysinecoated plates. Prepare plasmid dnalipid complexes recommend 2 doses of lipid. Lipofectamine 2000 transfection reagent is best transfection reagent available from life technologies for the co transfection of sirna and plasmid dna. May 11, 2016 the intracellular trafficking mechanism of lipofectaminebased transfection reagents and its implication for gene delivery. 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. Superior performance for co transfection of sirna and plasmid dna. Lipofectamine 3000efficient, reproducible transfection for.
Invitrogen lipofectamine 2000 transfection reagent. Methods h9c2 cells spread to 79 generation are cultivated in 6 well plate. Efficiency of dna transfection of rat heart myoblast cells. Three cell densities of hepg2 cells were transfected with gapd sirna 100nm using a range of volumes 0. Transfection protocol lipofectamine 3000 transfection reagent.
When the cell density reaches 9095%, transfecting three different target sequences plasmid paavzsgreenadrb11. Test serumfree media formulations for compatibility with lipofectamine. Mammalian cells transfection protocoljavier calvo follow invitrogen lipofectamine 2000 protocol. Boost lipofectamine2000 transfection efficiency with reduced cell toxicity. Transfection protocol lipofectamine 3000 transfection. After pei transfection of ppgk1egfp into h9c2 21 cells, egfp expression could be easily detected by fluorospectrometer after 48 h. Lipofectamine 2000 reagent protocol 202 lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. Several cell densities and lipid volumes were investigated to determine optimal transfection conditions, shown by the shaded boxes. I spoke to a technician who told me the best transfection reagent for my purposes is lipofectamine 3000, but my cells are not happy with the. 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. Posttranscriptional regulation of rbm5 expression in. American medical association, manual of style, 10th edition, 2007. Researchers use lipofectamine 2000 reagent for sirna and shrnabased gene knockdown experiments, as well as for gene expression studies.
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. The intracellular trafficking mechanism of lipofectamine. Transfection guide overview of transfection methods promega. Prepare transfection complexes by mixing 40 l of serum. Thee two most important factors in choosing transfecting reagent are. Add 100l of optimum to the eppendorf tube to dilute the dna and mix by tapping. Lipofectamine 2000 transfection protocol 10cm plates reagents. When h9c2 cells reached 85 % confluence, plasmids were mixed with lipofectamine 2000. Lipofectamine 3000 reagent provided higher gfp transfection efficiency than lipofectamine 2000 and fugene hd reagents for all five cell lines.
Twentyfour hours following appropriate plating, 12. Use a range of several ul of lipofectamine and ug dna to increase viability after transfection and efficiency. 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. H9c2 transfection reagent embryonic myocardium altogen. Maintain p19 cells undifferentiated in mem with 10% serum 7. Optimized conditions for successful transfection of human. 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. Research paper rac1 promotes the survival of h9c2 cells. Lipofectamine 2000 reagent protocol 202lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. This protocol is for transfecting plasmid dna into mammalian cells. Here, we report the results of a transfection trial with three promega reagents and another widely used transfection reagent. In tube b place 190 l of optimemi and 10 l lipofectamine 2000. Lipofectamine 2000 transfection reagent is versatile transfection reagent that has been shown to effectively transfect widest variety of adherent and suspension cell lines. Lipofectamine 2000 is a cationic liposome based reagent that provides high transfection efficiency and high levels of transgene expression in a range of.
Lipofectamine contains lipid subunits that can form liposomes in an aqueous environment, which entrap the. D1 transfection of h9c2 cells with the mir144 inhibitor had increased expression level of pten mrna in hypoxic conditions. Mix optimem and lipofectamine in another eppendorf 32 l lipofectamine in 0,8 ml optimem 3. Choosing the right transfection reagent for optimal efficiency. I use 24well plates for transfections in triplicates. Lipofectamine 2000 reagent is a lipidderived reagent designed to deliver exogenic nucleic acids dna andor rna into eukaryotic cells, either adherent or in suspension. Superior performance for cotransfection of sirna and plasmid dna. Transfection amounts component 96well 24well 6well dna per well 100 ng 500 ng 2500 ng p3000 reagent per well 0. Mix optimem with dna in an eppendorf 510 g dna0,8 ml optimem 2. Assayeach vector was transfected into h9c2 cells by using lipofectamine2000 invitrogen according to the manufacturers instructions. Cells from the h9c2 cell line express ach receptors and are often used as transfection hosts in the study of many myocardial diseases. Cut plasmid with an appropriate restriction enzyme to produce a linear fragment for transfection. Lipofectamine 2000 transfection reagent dartmouth college. 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.