This protocol is for transfecting plasmid dna into mammalian cells. 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. Invitrogen claims that this reagent gives the highest protein expression levels in the widest variety of adherent and suspension. Nih3t3 cells transfected for 2 hours with 6 l transpass p protein transfection reagent and 1 g igg labeled with a fluorescent dye a 549 fluorophore.
I use 24well plates for transfections in triplicates. Lipofectamine 2000 transfection protocol 10cm plates reagents. The media needs to be changed at least 1hr prior to transfection. Altogen cro offers in vivo rnai services, tumor xenograft models, toxicology testing, stable cell line generation, and. Theres a difference between the two protocols lipofectamine 3000 has an extra reagent that needs to be added. This reference provides a recommended procedure to transfect plasmid dna into huvec human umbilical vein endothelial primary isolates passage 0, cambrex, east rutherford, nj using lipofectamine. To perform transfection experiments in other cell culture plates, simply multiply the suggested quantities by the relative surface area of your plate. However, for cotransfection of sirnachimera and plasmid dna, lipofectamine 2000 should be used. The mechanism of lipofectamine 2000 mediated transmembrane. Lipofectamine is formulated to give high transfection efficiency with little toxicity to cells. Rnaimax transfection for huvec lipofectamine thermo fisher. Transfection, the introduction of nucleic acid into mammalian cells, is a powerful technique used to study in vivo gene function and regulation. A transfection complex mixture composed of 6 g of gfp expressing plasmid, 12 l of huvec reagent component and 25 l transpassv in 0.
The vector contained an ofp reporter gene and was transfected. Huvec sirna transfection help please sirna, microrna and rnai. The proprietary formulation of lipids and polymers ensures the highest possible transfection efficiencies and viabilities for huvec. The weight ratio of lipofectamine 2000 to pdna was kept constant at 3. Cytofect huvec transfection kit is a plasmid dna delivery system specifically optimized to deliver dna into huvec. In tube a place 2 g of dna in optimemi for a final volume of 200 l. Transfection of nih3t3 cells, hela, swis 3t3, 293t with. Sep, 2005 lipofectamine 2000 is an easy to use transfection reagent. In biontex experiments gfp positivity increased to 90. Protocol 1 stable transfection using lipofectamine 2000 in 6 well plates. Huvec sirna transfection help please sirna, microrna and. Content 3 formulation and storage 3 general considerations 3 transient huvec transfection 8 troubleshooting 12 ordering information.
Lipofectamine 2000 reagent thermo fisher scientific. This kit contains cytofect2, enhancer and media for transfection and culturing cells. Altogen biosystems provides in vivo transfection reagents, over 100 preoptimized in vitro transfection kits for cell lines and primary cells, and electroporation delivery products. Lipofectamine transfection of c2c12 cells bridges lab. Transient plasmid dna transfection into hela and hacat cells. Several cell densities and lipid volumes were investigated to determine optimal transfection conditions, shown by the shaded boxes. Comparative transfection efficiency of jetprime versus its main competitor. I have successfully form an intact and compact cell monolayer with huvec cells and now i want to apply the protocol of transfection. Invitrogen lipofectamine 3000 transfection reagent.
It is possible to plate and transfect the cells on the same day, but. Transfection was measured by luciferase activity using a conventional assay. Lipofectamine or lipofectamine 2000 is a common transfection reagent, produced and sold by invitrogen, used in molecular and cellular biology. I use 100pmol of sirna in 250ul optimem with 5ul of lipofectamine 2000 in 250ul of optimem.
Transfection reagents are highly efficient for dna and sirna transfection in vivo and in vitro. Huvec transfection optimized with cytofect kit youtube. Optimized conditions for successful transfection of human. Invitrogen lipofectamine 2000 transfection reagent fisher. Scaling up or down lipofectamine 3000 reagent transfections. Lipofectamine 2000 transfection reagent is versatile transfection reagent that has been shown to effectively transfect widest variety of adherent and suspension cell lines. I have successfully form an intact and compact cell monolayer with huvec cells and now i. Make sufficient 10% fbs in optimem media wo antibiotics sterile filter 2. Scaling up or down lipofectamine 3000 reagent transfections use the following table to scale the volumes for your transfection experiment. In tube b place 190 l of optimemi and 10 l lipofectamine 2000.
The cells were fixed, dapi stained and photographed 24 hours posttransfection. The same batch of the transfection reagent should be used for the whole experiment. Enable high transfection efficiency in novel genome editing applications. Researchers use lipofectamine 2000 reagent for sirna and shrnabased gene knockdown experiments, as well as for gene expression studies. Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. Do any one has protocol for stable transfection with lipofectamine 2000. Genscript recommends using lipofectamine 2000 for all transfections. Add 6l of lipofectamine2000 from biostores and 100 l of. Transfection protocol of lipofectamine rnaimax can be obtained from the invitrogen website. We observed a high batchtobatch variation in performance of the lipofectamine 2000 transfection reagent. It is possible to plate and transfect the cells on the same day, but cells must adhere to the bottom of the 6well dish. Prewarm 50ml of optimum stored in cold room at 4c 10min.
Tat peptide, plasmid dna pires2egfphgf and lipofectamine 2000 were used to prepare the tdl. Three cell densities of hepg2 cells were transfected with gapd sirna 100 nm using a range of volumes 0. Cells in a 6 well dish, plated and at 70% confluence. What percentage confluency should be used for stable transfection. I was wondering if someone have ever done such an experiment. Cut plasmid with an appropriate restriction enzyme to produce a linear fragment for transfection. Reverse transfection is the transfer of genetic material into cells and is reverse because the order of dna and cells is reverse that of conventional transfection. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. Highest transfection efficiency in many cell types and formats e. Transfecting plasmid dna into huvec cells using lipofectamine. Human umbilical vein endothelial cell huvec avalanche. Briefly, i feed my huvecs with fresh complete medium for 1 hour before transfection. Cells were transfected with a firefly luciferase encoding plasmid dna 0.
This kit contains cytofect2, enhancer and media for transfecting and culturing cells. Chok1, cos7, hela, hepg2, huh7, huvec, imr90, mcf7, mef, and nih3t3. Prepare transfection complexes by mixing 40 l of serum. Invitrogen lipofectamine 2000 transfection reagent. Too much lipofectamine will be too cytotoxic, and too little nucleic acid will give low efficiency of transfection. These are mixed as per the lipofecatmine 2000 protocol. Plate an amount that will result in approximately 85 to 90% confluence see table below on the day of transfection. Lipofectamine 2000 is tested for absence of microbial contamination with blood agar plates, sabaraud dextrose agar plates, and fluid thioglycolate medium, and functionally by transfection of chok1 cells with a reporter plasmid.
Lipofectamine 2000 transfection for hek293 cells about 6070% transfection rate transfect cells 6well plate. Reverse transfection on cell arrays for high content. Transient plasmid dna transfection into hela and hacat. Lipofectamine contains lipid subunits that can form liposomes in an aqueous environment. Lipofectamine 2000 transfection reagent from invitrogen. Promofectinhuvec cell transfection reagent instruction manual. Add 100l of optimum to the eppendorf tube to dilute the dna and mix by tapping.
Lipofectamine 2000 transfection, the expression of gfp was detected in 76,4% of hela cells with a mean fluorescence intensity of 437. I am about to start my stable transfections with lipofectamine 2000. Briefly, i feed my huvec s with fresh complete medium for 1 hour before transfection. Due to the broad range of maximal activity exhibited by lipofectamine rnaimax, a range of cell densities and volumes of lipofectamine rnaimax can be used for transfection. 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. B hela cells were transfected with luciferase encoding plasmid dna using either transit. Lipofectamine 3000 reagent maintains a high transfection efficiency within a robust dynamic range of lipid doses for quick and easy optimization. It is recommended that cells are growing at 9095% confluence prior to transfection for adherent cells.
A sample protocol is listed here for transfection experiments performed in 6well plates. What is nice about invitrogens lipofectamine 2000 is that it has an extensive, very useful manual which addresses these issues and helps tremendously cut down the initial time of protocol optimization. Lipofectamine 2000 transfection reagent dartmouth college. Transfection efficiency was assessed by facs analysis in various cell lines 24 h after transfection in 24well plates. The activity of delivered protein is maintained so active enzymes and other biomolecules such as antibodies can be directly delivered to cells without the need to obtain and transfect an expression construct. The aim of the present study was to explore the gene transfection efficiency of tat peptideplasmid dna liposome tdl compound combined with ultrasoundtargeted microbubble destruction utmd in human umbilical vein endothelial cell huvec. The proprietary formulation of lipids and polymers ensures the highest possible transfection efficiencies and viabilities for. Altogen cro offers in vivo rnai services, tumor xenograft models, toxicology testing, stable. Transfection efficiency of tdl compound in huvec enhanced. Transpass p is a protein transfection reagent that can deliver intact functional proteins and peptides with high efficiency into live mammalian cells via endocytosis. In hacat cells, lipofectamine 2000 achieved a transfection efficiency of 22. Lipofectamine 2000 reagent protocol 202lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart.
Cytofect huvec transfection kit the cytofect huvec transfection kit cat. The intracellular trafficking mechanism of lipofectamine. I wondering how much dna i should use for stable transfection with lipofectamine 2000 tranfection reagent. Optimizing transfection of primary human umbilical vein. 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. Tf200k is a plasmid dna delivery system specifically optimized to deliver dna into huvec.
Human umbilical vein endothelial cell huvec avalanche transfection reagent is a new, proprietary solution specifically designed for transfection on huvec. Of the nine reagents tested, lipofectamine 2000 and lipofectamine ltx demonstrated the highest transfection efficiency in huvec fig. Lipofectamine ltx reagent is a proprietary, animalorigin free formulation for the transfection of dna into eukaryotic cells with low cytotoxicity. Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations. Rnaimax transfection for huvec lipofectamine thermo. Conditions were used according to the manufacturers recommendation for both lipofectamine 2000 and for jetprime. Invitrogens lipofectamine 2000 is one of many cationic lipidbased transfection reagents presently on the market. In this context, since their launch in 1993, lipofectamine lfn reagents have become the most used trs, with over. It is used to increase the transfection efficiency of rna including mrna and sirna or plasmid dna into in vitro cell cultures by lipofection. Huvec transfection kit, umbilical vein endothelial cell. Lipofectamine 2000 reagent protocol 202 lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart.