Prepare 4 tubes each with 50 microliter of optimem medium, and label them 1 to 4. Optimizing transfection of primary human umbilical vein. Enhancing protein expression in hek293 cells by lowering. Many primary cell types have been successfully transfected with lipofectamine ltx reagent, including primary neural progenitor cells. 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. Plate cells so they will be 7090% confluent at the time of transfection. Invitrogen lipofectamine 3000 transfection reagent 1. Protocol for lipofectamine transfection in nih3t3 cells using cells plated in 8well plates. 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. May 11, 2016 the intracellular trafficking mechanism of lipofectaminebased transfection reagents and its implication for gene delivery. Dnalipofectamine ltx and plus complexes must be made in serumfree medium such as optimem. Such an enhancement is independent of transfection reagents, based on the test of calcium phosphate, lipofectamine 2000, lipofectamine ltx plus, and metafectene easy. Lipofectamine 2000 transfection for hek293 cells about 6070% transfection rate transfect cells 6well plate.
Pdf lipofectamine ltx is a cationic based transfection reagent that offers high levels of different transgenes expression in a huge number of. Invitrogen lipofectamine ltx reagent with plus reagent 1ml. Lipofectamine ltx with plus reagent thermo fisher scientific us. 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 transfection reagent from invitrogen. In this context, since their launch in 1993, lipofectamine lfn reagents have become the most used trs, with over 50,000 references to date. For transfecting thp1 cells should you use lipofectamine. For the cell lines easy to transfection, you can use either of the procedures reverse or forward.
This work was aimed to understand better the mechanism of this phenomenon and its potential. Lipofectamine ltx and plus reagent are proprietary formulations for transfecting nucleic acids into a wide range of eukaryotic cells. Transfection of 293 cells in 10 cm dishes abbreviated protocol surface area of 15 cm dish is 18 x greater than that of a 6well dish. Tell me about your experiences in transfection please, thanks. 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. How much lipofectamine plasmid is required for lentivirus production.
The instructions are recommending that you do this in duplicate with the two indicated different concentrations of lipofectamine 3000. The weight ratio of lipofectamine 2000 to pdna was kept constant at 3. Lipofectamine 3000 transfection reagent leverages our most advanced lipid nanoparticle technology to provide superior transfection performance with improved application outcomes and reproducible results. The intracellular trafficking mechanism of lipofectamine. Superior transfection reagents we were very happy and surprised to see lipofectamine 3000 reagent provide a more than 10fold difference in transfection ef.
Lipofectamine ltx reagent, when combined with plus reagent, provides the highest level of transfection in primary cells of any lipid or polymerbased reagent. Jun 10, 2011 during functional studies on the rat stressinducible hspa1b hsp70. 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. The day before transfection, seed 25,000 mcf7 cells per well of a 96well plate. Lipofectamine 2000 dna transfection reagent protocol see page 2 to view a typical dna transfection procedure. Efficient sirna delivery by the cationic liposome dotap in. 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. Lipofectamine transient transfection of cos7 cells to analyze. Higher densities can lead to spontaneous differentiation.
Make sufficient 10% fbs in optimem media wo antibiotics sterile filter 2. 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. Maintain p19 cells undifferentiated in mem with 10% serum 7. Theres a difference between the two protocols lipofectamine 3000 has an extra reagent that needs to be added. How much lipofectamine plasmid is required for lentivirus. Invitrogen lipofectamine 3000 transfection reagent. Invitrogen lipofectamine 3000 transfection reagent from.
However, for cotransfection of sirnachimera and plasmid dna, lipofectamine 2000 should be used. Lipofectamine or lipofectamine 2000 is a common transfection reagent, produced and sold by invitrogen, used in molecular and cellular biology. Transfecting sirna using lipofectamine raiax pin liu, 2017 declaration. Lipofectamine ltx reagent with plus reagent from thermo. Pdf optimizing transfection of umbilical cord mesenchymal stem. Dilute lipofectamine in serum and antibioticfree media in a different 15ml tube. Addition of the lipofectamine ltx plus reagent is a useful option to further increase the efficiency of. A new transfection reagent for effective transfection of primary cells, hardtotransfect cells and sensitive established cell lines. 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.
According to the manufacturers instructions, cells were grown overnight to reach a. The first one, lipofectamine ltx invitrogen transfection reagent, is based on. Combinational use of lipidbased reagents for efficient. Invitrogen lipofectamine ltx reagent with plus reagent. For additional information on scaling your transfection reaction, see page 4. Select the part name and then you can download the datasheet in pdf. For transfecting thp1 cells should you use lipofectamine 3000 or ltx. During functional studies on the rat stressinducible hspa1b hsp70. Dna lipofectamine ltx and plus complexes must be made in serumfree medium such as optimem. Intracellular delivery of biologically active proteins remains a formidable challenge in biomedical research. It is widely accepted that the final transfection efficiency te of lipidbased transfection reagents trs is ratelimited by several biological barriers such as cellular uptake, intracellular trafficking, endosomal escape and nuclear entry 1,2,3,4,5,6,7,8. We get 60% transfection efficiency gfp plasmid by using lipofectamine 3000 to transfect mcf7 cells. 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. Improve lentiviral production using lipofectamine 3000.
Lipofectmax 3000 transfection reagent abp biosciences. A new transfection reagent for effective transfection of. In the presence of plus reagent, lipofectamine ltx readily transfects. The histone demethylase kdm5bjarid1b plays a role in cell. If there appears to be largescale loss of cells after lipofectamine treatment, then it might pay to try using polylysinecoated plates. Lipofectamine ltx transfection reagent from invitrogen. Invitrogen lipofectamine 3000 transfection reagent can help improve lentivirus production. Scaling up or down lipofectamine 3000 reagent transfections. Taken together, all methods can be used for cell transfection but it always has to be tested. 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.
Sep 25, 2007 invitrogen have added a new product, lipofectamine ltx, to their extensive range of transfection products. Plasmids were transfected using lipofectamine ltx reagent with plus reagent in optimem according to the manufacturers instructions. Following on from other products in the lipofectamine range, lipofectamine ltx is a lipid based reagent for transfecting. Lipofectamine ltx reagent offers a new, advanced solution for gene expression studies in hardtotransfect cells. I have successfully nucleofected but would like to try transfection. Prepare 6 well plates by adding bakedsterile 22x22 1. You can generally use other serumfree medium instead of optimem. Lipofectamine ltx reagent is an advanced solution for gene expression in both common and ch. Lipofectamine ltx dna transfection reagents protocol. Comparative analysis of nonviral transfection methods in mouse. This work was aimed to understand better the mechanism of this phenomenon and its potential biological and. 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. Hek293 are pretty easy to transfect, you might want to test less fancy and less expensive. For this experiment, knockdown of endogenous luciferase was achieved in three engineered luciferase cell lines using lipofectamine.
Protocol for lipofectamine transfection in nih3t3 cells. The protocal is from thermo fisher scientific important guidelines for transfection 1. I personally have used this reagent to transfect cells for fluorescent microscopy, gst pull downs, and coimmunoprecipitation experiments. Laboratory 2010 lipofectamine 2000 transfection protocol 10cm plates reagents. 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. 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. 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. Lipofectamine 3000 reagent maintains a high transfection efficiency within a robust dynamic range of lipid doses for quick and easy optimization. I use 24well plates for transfections in triplicates. Lipofectamin ltx and plus reagent are highly effective transfection reagent but they are very expensive. Rna interference technology is an ideal strategy to elucidate the mechanisms associated with human hematopoietic stem cell differentiation into dendritic cells.
Lipofectamine 3000 protocol written out with timing. Pdf primary cells and certain established cells have generally been refractory to transfection using liposomesbased methods. Lipofectamine ltx reagent is advanced solution for gene expression in both common and challenging cell types. Lipofectamine ltx reagent with plus reagent efficiently transfects primary neural progenitor cells. Cells should be plated on collagencoated dishes, as for normal propagation. 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. Prepare transfection dilutions scaling the transfection considering the needs of the experiment. Addition of the lipofectamine ltx plus reagent is a useful option to further increase the efficiency of gene transfer. A method to evaluate the efficiency of transfection reagents in an. Culture plates were incubated at 37c and 5% co2 in shaking at 120 rpm. Lipofectmax 3000 transfection reagent is a lipidbased transfection reagent that forms a complex with dna or rna, and transports the complex into a. Transfection p19 cells with lipofectamine 2000 the same protocol can be used for 293t cells 1.
Pbs, trypsinized invitrogen, and disaggregated by manual pipetting to obtain a singlecell. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. We have had success using lipofectamine reagents in the past and the lipofectamine 3000 appears to be the best option. 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. Lipofectamine reagents go online to view related products. Transfection protocol of lipofectamine rnaimax can be obtained from the invitrogen website. Lipofectamine dna transfection reagents protocol see page 2 to view a typical plasmid transfection using a lipofectamine reagent. Liposomebased dna carriers may induce cellular stress. It is used to increase the transfection efficiency of rna including mrna and sirna or plasmid dna into in vitro cell cultures by lipofection. Yes, lipofectamine 3000 transfection reagent works well for delivery dna into mcf7 cells. Is it possible to use lipofectamine to transfect plasmid.
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