Make sufficient 10% fbs in optimem media wo antibiotics sterile filter 2. Superior transfection reagents we were very happy and surprised to see lipofectamine 3000 reagent provide a more than 10fold difference in transfection ef. Lipofectamine transient transfection of cos7 cells to analyze. Lipofectamine 2000 dna transfection reagent protocol see page 2 to view a typical dna transfection procedure. A method to evaluate the efficiency of transfection reagents in an. Lipofectamine ltx reagent is a plasmid transfection reagent that offers the right balance of potency and gentleness for your cells, resulting in the highest possible transfection efficiencies and viabilities. Select the part name and then you can download the datasheet in pdf. The histone demethylase kdm5bjarid1b plays a role in cell. The intracellular trafficking mechanism of lipofectamine. Dnalipofectamine ltx and plus complexes must be made in serumfree medium such as optimem. Yes, lipofectamine 3000 transfection reagent works well for delivery dna into mcf7 cells. For transfecting thp1 cells should you use lipofectamine. If there appears to be largescale loss of cells after lipofectamine treatment, then it might pay to try using polylysinecoated plates. Is it possible to use lipofectamine to transfect plasmid.
According to the manufacturers instructions, cells were grown overnight to reach a. Lipofectamine ltx reagent with plus reagent from thermo fisher scientific description lipofectamine ltx reagent is a plasmid transfection reagent that offers the right balance of potency and gentleness for your cells, resulting in the highest possible transfection efficiencies and viabilities. The day before transfection, seed 25,000 mcf7 cells per well of a 96well plate. Pdf optimizing transfection of umbilical cord mesenchymal stem. Lipofectamine reagents go online to view related products.
Lipofectamine ltx reagent, when combined with plus reagent, provides the highest level of transfection in primary cells of any lipid or polymerbased reagent. Higher densities can lead to spontaneous differentiation. In this context, since their launch in 1993, lipofectamine lfn reagents have become the most used trs, with over 50,000 references to date. 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. For this experiment, knockdown of endogenous luciferase was achieved in three engineered luciferase cell lines using lipofectamine. Transfection protocol of lipofectamine rnaimax can be obtained from the invitrogen website. Invitrogen lipofectamine 3000 transfection reagent. Scaling up or down lipofectamine 3000 reagent transfections. Tell me about your experiences in transfection please, thanks. I personally have used this reagent to transfect cells for fluorescent microscopy, gst pull downs, and coimmunoprecipitation experiments. A new transfection reagent for effective transfection of primary cells, hardtotransfect cells and sensitive established cell lines. Invitrogen lipofectamine 3000 transfection reagent 1. How much lipofectamine plasmid is required for lentivirus. Dilute lipofectamine in serum and antibioticfree media in a different 15ml tube.
Sep 25, 2007 invitrogen have added a new product, lipofectamine ltx, to their extensive range of transfection products. Dna lipofectamine ltx and plus complexes must be made in serumfree medium such as optimem. Plate cells so they will be 7090% confluent at the time of transfection. Maintain p19 cells undifferentiated in mem with 10% serum 7. Optimizing transfection of primary human umbilical vein. Here we show that biomedically relevant enzymes can be delivered into cells using a new dna transfection reagent, lipofectamine 3000, allowing assessment of their intracellular functions. Transfection p19 cells with lipofectamine 2000 the same protocol can be used for 293t cells 1. Lipofectmax 3000 transfection reagent is a lipidbased transfection reagent that forms a complex with dna or rna, and transports the complex into a. Combinational use of lipidbased reagents for efficient. The most efficient reagents were the cationic lipid reagents, lipofectamine 2000 and ltx, from invitrogen, which were able to genemodify up to 38% of huvec.
However, for cotransfection of sirnachimera and plasmid dna, lipofectamine 2000 should be used. Lipofectamine 2000 transfection reagent from invitrogen. Theres a difference between the two protocols lipofectamine 3000 has an extra reagent that needs to be added. Jun 10, 2011 during functional studies on the rat stressinducible hspa1b hsp70. We get 60% transfection efficiency gfp plasmid by using lipofectamine 3000 to transfect mcf7 cells.
Lipofectamine 3000 protocol written out with timing. In addition to being a top performing reagent for difficult cell types, invitrogen lipofectamine 3000 transfection reagent is a highly efficient, costeffective tool for lentiviral production. Protocol for lipofectamine transfection in nih3t3 cells using cells plated in 8well plates. This is the first time i will have used lipofectamine 3000, previously i only used lipofectamine 2000, and i am wondering if anyone has found an optimal lipo 3000 dna ratio for this cell line. Culture plates were incubated at 37c and 5% co2 in shaking at 120 rpm. That said, lipofectamine 2000 seems to be a better reagent when a lower amount of the cdna is used for transfection for gfp this amount can be as low as 0. Mar 16, 2018 in conclusion, we present a simple method for ef ficient gene transfer in primary fibroblasts and hepatoblasts based on the combined use of lipofectamine ltx and fugene hd. Scaling up or down lipofectamine 3000 reagent transfections use the following table to scale the volumes for your transfection experiment. Many primary cell types have been successfully transfected with lipofectamine ltx reagent, including primary neural progenitor cells. This work was aimed to understand better the mechanism of this phenomenon and its potential biological and. This work was aimed to understand better the mechanism of this phenomenon and its potential. Such an enhancement is independent of transfection reagents, based on the test of calcium phosphate, lipofectamine 2000, lipofectamine ltx plus, and metafectene easy.
Two complementary approaches for intracellular delivery of. Prepare transfection dilutions scaling the transfection considering the needs of the experiment. For the cell lines easy to transfection, you can use either of the procedures reverse or forward. Transfecting sirna using lipofectamine raiax pin liu, 2017 declaration. The weight ratio of lipofectamine 2000 to pdna was kept constant at 3. Lipofectamine ltx reagent with plus reagent efficiently transfects primary neural progenitor cells. Lipofectamin ltx and plus reagent are highly effective transfection reagent but they are very expensive. Prepare 6 well plates by adding bakedsterile 22x22 1. Lipofectamine or lipofectamine 2000 is a common transfection reagent, produced and sold by invitrogen, used in molecular and cellular biology.
Lipofectmax 3000 transfection reagent abp biosciences. In conclusion, we present a simple method for ef ficient gene transfer in primary fibroblasts and hepatoblasts based on the combined use of lipofectamine ltx and fugene hd. Efficient sirna delivery by the cationic liposome dotap in. Invitrogen lipofectamine 3000 transfection reagent from. Cells should be plated on collagencoated dishes, as for normal propagation. Volume glutamax gibco 35050 5 ml penicillinstreptomycin gibco 15140122 5 ml fbs gibco 26140079 50 ml 10% final dmem gibco 11965084 fill to 500 ml use a 0.
It is used to increase the transfection efficiency of rna including mrna and sirna or plasmid dna into in vitro cell cultures by lipofection. Lipofectamine 3000 reagent maintains a high transfection efficiency within a robust dynamic range of lipid doses for quick and easy optimization. Lipofectamine dna transfection reagents protocol see page 2 to view a typical plasmid transfection using a lipofectamine reagent. Amaxa electroporation different cell lines give different transfection rates at different seeding conditions and exposure time serum free with serum, dnalipid concentration with ltx, you can also try plus, nupherin, combimag, etc. Comparative analysis of nonviral transfection methods in mouse. Liposomebased dna carriers may induce cellular stress. Lipofectamine ltx and plus reagent are proprietary formulations for transfecting nucleic acids into a wide range of eukaryotic cells. Invitrogen lipofectamine ltx reagent with plus reagent. In the presence of plus reagent, lipofectamine ltx readily transfects. You can generally use other serumfree medium instead of optimem. Lipofectamine ltx reagent with plus reagent from thermo. Lipofectamine 3000 transfection reagent leverages our most advanced lipid nanoparticle technology to provide superior transfection performance with improved application outcomes and reproducible results. We have had success using lipofectamine reagents in the past and the lipofectamine 3000 appears to be the best option. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines.
Protocol for lipofectamine transfection in nih3t3 cells. Laboratory 2010 lipofectamine 2000 transfection protocol 10cm plates reagents. Invitrogen lipofectamine ltx reagent with plus reagent 1ml. For additional information on scaling your transfection reaction, see page 4. May 11, 2016 the intracellular trafficking mechanism of lipofectaminebased transfection reagents and its implication for gene delivery. Taken together, all methods can be used for cell transfection but it always has to be tested. Improve lentiviral production using lipofectamine 3000. During functional studies on the rat stressinducible hspa1b hsp70. For transfecting thp1 cells should you use lipofectamine 3000 or ltx. I have successfully nucleofected but would like to try transfection. Pdf lipofectamine ltx is a cationic based transfection reagent that offers high levels of different transgenes expression in a huge number of. Enhancing protein expression in hek293 cells by lowering. To transfect cells in different tissue culture formats, vary the amounts of dna, lipofectamine ltx, plus reagent cells, and medium used in proportion to the relative surface area, as shown in the table below and given on a per well basis.
Lipofectamine 3000 protocol written out with timing specifics. Our study demonstrated that a small selection of commercially available chemical transfection reagents was able to transfer exogenous genes efficiently to primary human cells. Dilute lipofectamine3000 reagent in optimem medium 2 tubes, mix well follow the instructions as indicated. Simple manipulations in vitro can unequivocally yield alloreactive or tolerogenic populations, suggesting key implications of biochemical players that might emerge as therapeutic targets for cancer or graftversushost disease. 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 ltx dna transfection reagents protocol. Hek293 are pretty easy to transfect, you might want to test less fancy and less expensive. Lipofectamine ltx transfection reagent from invitrogen. The first one, lipofectamine ltx invitrogen transfection reagent, is based on. Plasmids were transfected using lipofectamine ltx reagent with plus reagent in optimem according to the manufacturers instructions. Prepare 4 tubes each with 50 microliter of optimem medium, and label them 1 to 4. Lipofectamine ltx with plus reagent thermo fisher scientific us. Addition of the lipofectamine ltx plus reagent is a useful option to further increase the efficiency of.
Pbs, trypsinized invitrogen, and disaggregated by manual pipetting to obtain a singlecell. No other plasmid dnaspecific transfection reagent can match efficiency, convenience, and gentleness of lipofectamine ltx reagent for transfection of primary, challenging, and sensitive cell lines. Pdf primary cells and certain established cells have generally been refractory to transfection using liposomesbased methods. Lipofectamine 2000 transfection for hek293 cells about 6070% transfection rate transfect cells 6well plate. Qc specifications back to table of content lipofectamine 2000 is tested for the absence of microbial contamination using blood agar plates, sabaraud dextrose agar plates, and fluid thioglycolate medium, and functionally by transfection of chok1 cells with a reporter plasmid. The intracellular trafficking mechanism of lipofectaminebased transfection reagents and its implication for gene delivery. Lipofectamine ltx reagent is advanced solution for gene expression in both common and challenging cell types. Lipofectamine ltx reagent offers a new, advanced solution for gene expression studies in hardtotransfect cells. Following on from other products in the lipofectamine range, lipofectamine ltx is a lipid based reagent for transfecting. The protocal is from thermo fisher scientific important guidelines for transfection 1. The instructions are recommending that you do this in duplicate with the two indicated different concentrations of lipofectamine 3000.
1445 216 800 1323 1219 712 863 999 171 664 527 682 524 673 1034 227 1473 301 1160 1275 488 1380 935 310 175 233 936