Tell me about your experiences in transfection please, thanks. We employed lipofectamine to transfect the cells and compared efficiency by using the reagents lipofectamine 2000, 3000, and ltx. Lipofectamine or lipofectamine 2000 is a common transfection reagent, produced and sold by invitrogen, used in molecular and cellular biology. I got it to work very well before christmas but it hasnt worked since because the transfection efficiency has been very low.
The following cell lines have been successfully transfected with the indicated transfection reagents. Invitrogen lipofectamine ltx reagent with plus reagent 1ml. Enhancing protein expression in hek293 cells by lowering. Highest transfection efficiency in many cell types and formats e. Despite this, a satisfactory mechanismbased explanation of their. Lipofectamine dna transfection reagents protocol see page 2 to view a typical plasmid transfection using a lipofectamine reagent. The transfection performance of cellspecific dnain reagents were compared to those of lipofectamine 2000 and lipofectamine 3000 lf3k. 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. Information and translations of lipofectamine in the most comprehensive dictionary definitions resource on the web.
May 11, 2016 lipofectamine reagents are widely accepted as goldstandard for the safe delivery of exogenous dna or rna into cells. Overexpression of g induces neurite outgrowth in pc12 cells. Lipofectamine ltx was identified as the optimal transfection reagent as a result of its higher transfection efficiency at shorter periods of time following transfection when cytotoxicity was limited. They have collected protocols for many cell lines and created. Lipofectamine ltx reagent is a plasmid transfection reagent that offers a balance of potency and gentleness for your cells, resulting in high transfection efficiencies and viabilities. Select the part name and then you can download the datasheet in pdf. Lipofectamine ltx reagent, in the presence of plus reagent, provided higher transfection efficiency and gfp expression than reagents f6 and fhd.
Derivation and characterization of primordial germ cells from. 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 dna transfection reagent protocol see page 2 to view a typical dna transfection procedure. Lipofectamine 3000 reagent maintains a high transfection efficiency within a robust dynamic range of lipid doses for quick and easy optimization. Lipofectamine ltx may be used with or without serum which adds to its flexibility. Im having problems transfecting my raw cells using lipofectamine or lipofectamine ltx. 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. L of lipofectamineltx was then added and allowed to complex at room temperature. Invitrogen lipofectamine 3000 transfection reagent. Transfection of 293 cells in 10 cm dishes abbreviated protocol surface area of 10 cm dish is 8 x greater than that of a 6well dish. They have collected protocols for many cell lines and created an excellent reference database for the tissue culture scientist. For additional information on scaling your transfection reaction, see page 4. Lipofectamine ltx transfection reagent from invitrogen.
Lipofectamine ltx reagent, with plus reagent, efficiently transfects primary neural progenitor cells. Mix gently and incubate for 515 minutes at room temperature. Nerve growth factor induces neurite outgrowth of pc12. Lipofectamine 2000 transfection reagent dartmouth college. Follow manufacturers handbook protocol unless otherwise indicated. A new transfection reagent for effective transfection of. This work was aimed to understand better the mechanism of this phenomenon and its potential. L of dna, plus reagent and optimem combination for each transfection replicate. Invitrogen lipofectamine ltx reagent with plus reagent. Enable high transfection efficiency in novel genome editing applications. Lipofectamine 2000 transfection for hek293 cells about 6070% transfection rate transfect cells 6well plate. Optimizing the generation of stable neuronal cell lines via. Invitrogen lipofectamine 3000 transfection reagent from. Lipofectamine transient transfection of cos7 cells to.
In the presence of plus reagent, lipofectamine ltx readily transfects plasmids into cells such as raw 267. This reagent provides an advanced solution for gene expression in common cell lines, particularly chinese hampster ovary cho, and challenging cell types such. Transfection using effectene, lipofectamine, and polyfect reagents many reagents are now available for optimization of cell transfection efficiency. For transfecting thp1 cells should you use lipofectamine 3000 or ltx. In the presence of plus reagent, lipofectamine ltx readily transfects. Mcf7 cells were cotransfected with firefly luciferase reporter, renilla luciferase plasmid, and the indicated expression constructs, in a final volume of 0. Prepare transfection dilutions scaling the transfection considering the needs of the experiment. Lipofectamine ltx and plus reagent are proprietary formulations for transfecting nucleic acids into a wide range of eukaryotic cells.
Dna lipofectamine ltx and plus complexes must be made in serumfree medium such as optimem. Scaling up or down lipofectamine 3000 reagent transfections use the following table to scale the volumes for your transfection experiment. Prepare 6 well plates by adding bakedsterile 22x22 1. It can be used for transfection of adherent or suspension cells with dna or rna including stealth rnai and sirna and can be used for either transient transfections or to create stable cell lines. Target delivery of a gene into the brain using the rvg29. It is worthwhile taking a look at the invitrogen transfection website as the support offered is impressive. Zeb1nurd complex suppresses tbc1d2b to stimulate e.
Total master mix volume should have 1100 ulplate with a little. Lipofectamine reagents are widely accepted as goldstandard for the safe delivery of exogenous dna or rna into cells. Lipofectamine ltx reagent with plus reagent efficiently transfects primary neural progenitor cells. L of lipofectamineltx was then added and allowed to complex at room temperature for 90 min. Lipofectamine 2000 is a lipid based transfection reagent for eukaryotic cells. Combine diluted dna and lipofectamine, mix and incubate at room temperature for 20 min. Dnalipofectamine ltx and plus complexes must be made in serumfree medium such as optimem. While diluting the dna, warm lipofectamine ltx to room temperature. Plate cells so they will be 7090% confluent at the time of transfection. Optimizing transfection of primary human umbilical vein. Dilute lipofectamine in serum and antibioticfree media in a different 15ml tube.
Lipofectamine 2000 reagent thermo fisher scientific. Lipofectamine ltx reagent is advanced solution for gene expression in both common and challenging cell types. The firefly and renilla luciferase activities were assayed using dual luciferase assays promega after 48 h transfection. As well as suggesting an ultra version of lipofectamine such as lipofectamine ltx or plus, you could check that your detection method for detecting an efficient transfection is working properly. If you let them reach confluency, even if this happened a few passages ago, the transfection efficiency will be significantly diminished. Effi ciency and ofp expression were analyzed 72 hours posttransfection and a u2os and b hepg2 cells showed 4fold and 80fold improvement with lipofectamine. Lipofectamine ltx dna transfection reagents protocol. Highest transfection efficiency was seen with lipofectamine 2000, followed by ltx. Pdf primary cells and certain established cells have generally been refractory to transfection using liposomesbased methods. Lipofectamine reagents go online to view related products. We appreciate greatly the technical service of thermofisher and find them an. Product is guaranteed for 6 months from the date of shipment if stored properly. Mix dna, plus reagent invitrogen and optimem combination well. Im not sure what your endpoint is for the successful transfection e.
Lipofectamine 2000 transfection reagent from invitrogen. Mar 08, 2008 im having problems transfecting my raw cells using lipofectamine or lipofectamine ltx. Dna lg and plus reagent ll varied with xtremegene hp, the volume of transfection mix added to the wells. For several of these cells, dnain surpassed lipofectamine i i is performance figure i. Sep 25, 2007 lipofectamine ltx may be used with or without serum which adds to its flexibility.
Derivation and characterization of primordial germ cells. It is used to increase the transfection efficiency of rna including mrna and sirna or plasmid dna into in vitro cell cultures by lipofection. Different types of transfection reagents can be used for transfection, optimize the amounts of dna and liposomes for each type. Lipofectamine ltx in the presence of plus reagent is an excellent choice for transfection of human cancer cell lines as well as primary and stem cells. Combinational use of lipidbased reagents for efficient transfection. Still, the overall numbers of available zebrafish cell lines and. For transfecting thp1 cells should you use lipofectamine. With a p tip scratch off a small amount of frozen bac and mix for each inoculation tube. Refer to the cell lines database for a list of cell types successfully transfected. Lipofectamine transient transfection of cos7 cells to analyze. One of the oldest methods is transfection using diethylaminoethyldextran deaed. Lipofectamine 3000 provided gentle but efficient transfection in several line of interest to our lab, heioc1 mouse cochlear 69fold, nccit and ntera2 3 fold testicular cancer lines and allowed for very nice dose response assays for cisplatin sensitvity assays. Forkhead box protein k1 recruits tet1 to act as a tumor.
Lipofectamine contains lipid subunits that can form liposomes in an aqueous environment, which entrap the. The vector contained an ofp reporter gene and was transfected with lipofectamine 2000 or lipofectamine 3000 reagent into a u2os and b hepg2 cell lines. I also saw, for the first time ever, true 100% transfection efficiency of hek293 cells. Jun 10, 2011 during functional studies on the rat stressinducible hspa1b hsp70. Invitrogen lipofectamine 3000 transfection reagent 0.
Protocol for dna transfection and luciferase assay using pfr. Pc12 cells were cotransfected with yfptagged constructs encoding a g. Scaling up or down lipofectamine 3000 reagent transfections. It increases the likelihood of successful cleavage and recombination with talens or crisprs through highly efficient transfection, ultimately maximizing the efficiency of genetic modifications and simplifying the downstream processes. 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. As described in the manufacturers instructions, the gene transfer efficiency of lipofectamine ltx is increased by the addition of plus reagent to. I agree with parvoman that one of the main considerations is how well the cells are growing. Lipofectamine 2000 and lipofectamine ltx invitrogen, carlsbad, ca, usa gave the highest transfection efficiencies of the reagents tested. Lipofectamine 3000 reagent is suitable for novel genome editing applications. Product is guaranteed for 6 months from the date of shipment if. Plasmids were transfected using lipofectamine ltx reagent with plus reagent in optimem according to the manufacturers instructions. I have successfully nucleofected but would like to try transfection. 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.
Protocol for dna transfection and luciferase assay using. A method to evaluate the efficiency of transfection reagents in an. This work was aimed to understand better the mechanism of this phenomenon and its potential biological and. During functional studies on the rat stressinducible hspa1b hsp70. Then add an appropriate volume of lipofectamine to each plasmid master mix. 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. Transfections were performed using lipofectamineltx invitrogen. The intracellular trafficking mechanism of lipofectamine. Jul 17, 2007 lipofectamine 2000 is a lipid based transfection reagent for eukaryotic cells.
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