Lipofectamine 2000 transfection, the expression of gfp was detected in 76,4% of hela cells with a mean fluorescence intensity of 437. Mdck, madindarby canine kidney, sirna, lipofectamine rnaimax, 778. Nonviral, transient transfection of plasmid dna into differentiated, polar epithelia. Prewarm 50ml of optimum stored in cold room at 4c 10min. Optimized conditions for successful transfection of human. Transient plasmid dna transfection into hela and hacat cells. Lipofectamine transfection of c2c12 cells bridges lab.
Invitrogen lipofectamine 2000 transfection reagent strong performance in a wide range of cell lines reliableone of the mostcited dna reagents strong performancefor easytotransfect to moderately difficulttotransfect cell lines easy to usesimple protocol for easy optimization in a. Prepare the transfection buffer as noted in the material section. Lipofectamine reagents are widely accepted as goldstandard for the safe delivery of exogenous dna or rna into cells. Lipofectamine transient transfection of cos7 cells to. Genscript recommends using lipofectamine 2000 for all transfections. The transfection protocol for lipofectamine 3000 reagent was developed to be easy to use while still ensuring optimum performance and reliability in a wide panel of cell lines. Research open access the interaction of celecoxib with. Invitrogen lipofectamine 2000 transfection reagent fisher. Lipofectamine 2000 reagent delivers dna or sirna with excellent transfection performance for protein expression, gene silencing, and functional assays. Ask for a sample a comparison showing transfection efficiency of polyjet reagent vs. Lipofectamine 2000 right panel around 5% efficiency. Add 100l of optimum to the eppendorf tube to dilute the dna and mix by tapping. How does transfection with lipofectamine 2000 work.
Aquaporin2 transfection of madindarby canine kidney cells reconstitutes vasopressinregulated transcellular osmotic water transport. Make sure and evenly distribute the cells on the coverslips by swirling plate. A schematic representation of the protocol is illustrated in figure 3. Be sure to mix reagent invert 5 to 10 times prior to taking an aliquot for transfection. Higher densities can lead to spontaneous differentiation. In tube a place 2 g of dna in optimemi for a final volume of 200 l. Concerning mdck, dreamfect gold is really efficient for transfection and also co transfection as demonstrated for example by zhang y. Plasmid was made up in lipofectamine or lipofectamine 3000 thermo. Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations. 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 mechanism of lipofectamine 2000 mediated transmembrane. Transfection was measured by luciferase activity using a conventional assay. Despite this, a satisfactory mechanismbased explanation of their. Transfection protocol lipofectamine 3000 transfection. A sample protocol is listed here for transfection experiments performed in 6well plates. Conversion of mdck cell line to suspension culture by. Mdck transfection kit includes mdck transfection reagent 0. Each reagent was used to transfect hek 293, hela, lncap, a549, and hepg2 cells in 96well format. Lipofectamine 2000 are the most efficacious and showed little or no.
Transfection of mcf7 with lipofectamine 2000 maartje vogel. I have been attempting to transfect my mdck cells for a while now. Lipofectamine 2000 transitx2 lipofectamine 2000 gapdh aha1 transfection reagent sirna target transitx2 dynamic delivery system continued 90 80 70 60 50 40 30 0 egfp positive % 20 10 a549 chok1 mdck lncap hmec 100 hep g2 pc12 nhdf transitx2 mirus bio was used to transfect plasmid dna encoding egfp into the indicated cell lines. Thermo scientific nunc 24well cellculture treated multidishes 142475 invitrogen lipofectamine 3000 transfection reagent l3000008 gibco optimem i reduced serum medium 31985062 step tube complexation components amount per well 24well.
Adhesion receptors mediate efficient nonviral gene delivery. Invitrogen lipofectamine 2000 transfection reagent life. Plate an amount that will result in approximately 85 to 90% confluence see table below on the day of transfection. The firstgeneration lipofection was a singlecomponent system, in which only the lipid nl. A comparison showing transfection efficiency of polyjet reagent vs. For cellfectin dmriec, and lipofectamine 2000 reagents this is normal. Evidently, upon loss of cellcell contact between mdck cells, lipoplex binding. The following transfection and infection protocol is established for 6wellplates.
For generation of stable transfectants, mdck cells were transfected with the hahpept1pcdna3 plasmid using lipofectamine 2000 reagent, and were grown in 0. Transfection reagent a division of gene therapy systems, inc. Each reagent was used to transfect hek 293, hela, lncap, a549, and hepg2 cells in a 96well format, and. The intracellular trafficking mechanism of lipofectamine. Sep 01, 2009 the transfection procedure was as follows. In biontex experiments gfp positivity increased to 90.
It is possible to plate and transfect the cells on the same day, but cells must adhere to the bottom of the 6well dish. Transfection p19 cells with lipofectamine 2000 the same protocol can be used for 293t cells 1. Gfp expression was assayed by fluorescence microscopy in verocells 24 h after transfection. This compound is commercially available from different companies either as a non. The madindarby canine kidney cell line mdck ii was cultured in 75 cm. Protocol 6 well dish plate cells and grow to 70% confluence in 2 ml media without antibiotics dmem with 10% fbs, no psg. Add 6l of lipofectamine2000 from biostores and 100 l of. Pool dishes of cells that have been prepared for transfection. 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 2000 reagent thermo fisher scientific. Lipofectamine 3000 can be used to transfect a broad spectrum of cell line with nucleic acids. The above insertion was verified by dna sequencing. Transfection efficiency was assessed by facs analysis in various cell lines 24 h after transfection in 24well plates.
Highest transfection efficiency in many cell types and formats e. Improving transient transfection efficiency in a differentiated, polar epithelial. Cellspecific transfection protocols thermo fisher scientific us. Anyone familiar with transfection of mdck epithelial cells using. This result shows that simple, lipidmediated transfection of hep g2 cells is a viable alternative to gene.
Transfection of cells with plasmid dna is well optimized, and the procedure is simple and quick. Transit2020 transfection reagentfor hard to transfect cell types. It is a relatively modern method first described by felgner et al. 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 transfection reagent is a proprietary formulation for the transfection of nucleic acids dna and rna into eukaryotic cells and provides the following advantages. Transient transfection of 293t cells faculty websites. Conversion of mdck cell line to suspension culture.
Transfection reagents are highly efficient for dna and sirna transfection in vivo and in vitro. With proprietary shaved cell transfection protocol, polyjet left panel gives up to 70% gfp positive cells vs. Gibco hams f12 nutrient mix 11765054 5% horse serum 1 mm gibco sodium pyruvate 160070 25 mm gibco hepes 15630106 proper culture techniques and procedures are an essential. Transfection efficiency of p19 cells is between 3050% from 5099% for many other stable cell lines.
In tube b place 190 l of optimemi and 10 l lipofectamine 2000. The plasmid dna was introduced into umuc3 cells using lipofectamine ltx invitrogen. L lipofectamine 2000 and incubated together with the cells in optimem i medium invitrogen for 4 h. It is best to use polypropylene instead of polystyrene tubes. For all transfection of multiple wells in 6 well plate, multiply cdna, optimem, and lipofectamine quantity used by number of wells to transfect. It is possible to plate and transfect the cells on the same day, but cells must adhere to. Resuspend at 2 gml, and take the od on a small sample a 1. Hek293ft cells were transfected with gfp vector pegfpn3 by polyjet left panel and lipofectamine 2000 right panel respectively. Transfection of nih3t3 cells, hela, swis 3t3, 293t with. To optimize the amount of lipofectamine 2000 for transfection in a 24well plate, start. A highly efficient method of transfecting hep g2 cells. Optimemin ul, equal volume for dna and lipofectamine lipofectamine in ul 25. A highly efficient method of transfecting nih3t3 cells. Lipofectamine 2000 transfection reagent dartmouth college.
Co transfection of multiple plasmid dnas is a technique increasingly employed by biology researchers. In search for a cheap and effective transfection reagent we used the positively charged polyplex. The products included in this piece are our most robust transfection reagents that provide solutions to both common and hardtotransfect cell types, promoting effective. 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. For additional information on scaling your transfection reaction, see page 4.
Prepare plasmid dnalipid complexes recommend 2 doses of lipid. High efficiency transfection reagent at a reasonable price. Transfection protocol transfection components component cat. All other chemicals were products of commercial biological grade and were obtained from. Also prepare the sterile dna for transfection 2030 g10cm dish70 for a 15cm dish. B hela cells were transfected with luciferase encoding plasmid dna using either transit. Lipofectamine 2000 cd reagent is a proprietary animal originfree formulation for transfecting nucleic acids into eukaryotic cells. Of all transfection reagents, lipofectamine 2000 enables you to. Improving transient transfection efficiency in a differentiated, polar.
Laboratory 2010 lipofectamine 2000 transfection protocol 10cm plates reagents. Lipofectamine 2000 cd reagent complexes can be added directly to cells in culture medium. Transfection efficiency was assessed by facsanalysis 24 h after transfection of plasmid dna encoding for egfp pcmvegfp with jetoptimus, lipofectamine 2000 and lipofectamine 3000 in primary cells and cancer cell lines. Generation of recombinant influenza virus from plasmid dna. Cell lines transfection the psg5cox2 plasmid, which contains a fulllength human cox2 cdna in the psg expression vector 23, was used for cox2 transfection. To transfect cells with sirna, follow the protocol as. Invitrogen lipofectamine transfection reagent protocols have been. However, for cotransfection of sirnachimera and plasmid dna, lipofectamine 2000 should be used. Lipofectamine 2000 reagent protocol 202lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. Comparative transfection efficiency of jetprime versus its main competitor. Transfection protocol of lipofectamine rnaimax can be obtained from the invitrogen website. Researchers use lipofectamine 2000 reagent for sirna and shrnabased gene knockdown experiments, as well as for gene expression studies. Transfection transient transfection of hek cells was performed as described 7.
The weight ratio of lipofectamine 2000 to pdna was kept constant at 3. Anyone familiar with transfection of mdck epithelial cells. To perform transfection experiments in other cell culture plates, simply multiply the suggested quantities by the relative surface area of your plate. A few applications that popularly utilize co transfection are virus production, proteinprotein interaction studies, stable cell line generation, or simple addition of reporter dna constructs to normalize experimental output. Lipofectamine 2000 reagent can be used for transfection of plasmid dna and small rnas. Max % of positive cells after transfection of a gfpplasmid 3. At the foundation of the popularity of this broadspectrum reagent is the reliably high efficiency and simple protocol for success across a broad range of cell lines figure 1. Lipofectamine 2000 transfection for hek293 cells about 6070% transfection rate transfect cells 6well plate. Plate cells so they will be 7090% confluent at the time of transfection.
This new reagent is able to deliver plasmid dna into the fibroblast cell line, nih3t3, with three fold greater efficiency than the popular commercial transfection reagent, lipofectamine 2000 lf2k. Examples of cells transfected successfully thermo fisher. Altogen cro offers in vivo rnai services, tumor xenograft models, toxicology testing, stable cell line generation, and. Arpe19 transfection kit retinal pigment epithelium. L reverse transfection of rnai reverse transfection is faster to perform than forward transfection and is the method of choice for highthroughput transfection. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. Maintain p19 cells undifferentiated in mem with 10% serum 7.
Transfection protocol lipofectamine 3000 transfection reagent mcf10a breast cancer cells complete growth medium component cat. In hacat cells, lipofectamine 2000 achieved a transfection efficiency of 22. I am using lipofectamine 2000 reagent from life technologies. Lipofection has become more common than the capo 4 precipitation, because it is just as simple to use but frequently provides a much better transfection efficiency. Efficient electroporation of dna and protein into confluent and. Lncap, mdck, pc12, primary human mammary epithelial cells hmec and. Lipofectamine transient transfection of cos7 cells to analyze. Invitrogen lipofectamine 2000 transfection reagent. Mdck transfection reagent is supplied in liquid form at a concentration of 0. The reagent provides high transfection efficiency in many cells types and formats. I use 24well plates for transfections in triplicates.
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