Lipofectamine 2000sirna complexes cause endoplasmic reticulum. Lwell of lipofectamine 2000 and dharmafect 4 transfection reagents. Content 3 formulation and storage 3 general considerations 3 transient huvec transfection 8 troubleshooting 12. Maintain p19 cells undifferentiated in mem with 10% serum 7. Transfection p19 cells with lipofectamine 2000 the same protocol can be used for 293t cells 1. Refer to the cell lines database for a list of cell types successfully. Upregulation of microrna17 aggravates vascular endothelial. Lipofectamine 2000 reagent protocol 202 lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. Lipofection is not very effective in transfecting huvec that kind of slowing growing. The impact of transfection mediated toxicity gene expression and cytotoxicity analysis of transfection reagents introduction while plasmid dna delivery is a widely used method to study cellular functions of proteins of interest, studies to identify nontoxic gene. High efficiency transient transfection of genes in human.
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 human umbilical vein endothelial cell line huvec is a powerful tool in the study of various physiological processes such as angiogenesis. The reagent provides high transfection efficiency in many cells types and formats. Prepare plasmid dnalipid complexes recommend 2 doses of lipid. Plate cells so they will be 7090% confluent at the time of transfection. The best way was lipofectamine 2000 and of course the electroporation using. Highest transfection efficiency in many cell types and formats e. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. To transfect cells with sirna, follow the protocol as. High efficiency transient transfection of genes in human umbilical vein endothelial cells by electroporation. I use 24well plates for transfections in triplicates. Abstract lipofectamine 2000 lipo2000 delivery system is commonly used for short interfering rna sirna transfection, whereas the cellular responses. Lipofectamine 2000 invitrogen, paisley, uk and com plexes were prepared following the manufacturers recom. Huvec cells at 70% confluency were transfected in optimem medium invitrogen, carlsbad, ca with the indicated sirna duplexes using lipofectamine 2000 invitrogen.
If you wish to use antibiotics during transfection, test your conditions thoroughly. The proprietary formulation of lipids and polymers ensures the highest possible transfection efficiencies and viabilities for huvec. Improved transfection of huvec and mef cells using dna indian. Primary cells, such as huvec, are notoriously difficult to transfect and are. Culture vessel multiplication factor shared reagents dna transfection sirna transfection growth medium optimem medium for complexing dna p3000 reagent lipofectamine. Three cell densities of hepg2 cells were transfected with gapd sirna 100nm using a range of volumes 0. Original article an improved method for increasing the efficiency of. For determination which of the two nucleofector tm kits is best suited for efficient transfection of a specific human stem cell clone in nucleofector tm iii2b device. Lipofectamine 2000 reagent thermo fisher scientific.
What is the best protocol for transfection into huvec. Ii am trying to transfect huvec, i have no exptience in transfection, i will make transfection for enos gene, what is the best protocol for transfection and after that what is the best method to. For the cell lines easy to transfection, you can use either of the procedures reverse or forward. The best way was lipofectamine 2000 and of course the electroporation. Lipofection, also known as lipid transfection or liposomebased transfection, uses a lipid complex to deliver dna to cells. Huvec transfection kit, umbilical vein endothelial cell. 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. Transfection efficiency and fluorescence optical analyses the in vitro transfection efficiency of lipofectaminetm 2000 invitrogen was evaluated in hne cells using the pegfpc1 plasmid clontechtakara bio europe, saintgermainenlaye, france. The huvec cell line was derived and established from human umbilical vein endothelial cells, which is also where it gets its name. Co transfection of pegfpc1 and gfp sirna and h 2o transfections were used as controls. Promofectin huvec cell transfection reagent instruction. Optimizing transfection of primary human umbilical vein. Description based on our proprietary polymer synthesis technology, polyjet dna in vitro transfection reagent is formulated as a biodegradable polymer based dna transfection reagent that ensures effective and reproducible transfection on hek293, cos7, nih3t3, hela, cho and a broad ranges of hardtotransfect. Lipofectamine 2000 transfection for hek293 cells about 6070% transfection rate transfect cells 6well plate.
Hi, i tried to transfect my hek293t cells with pegfp using lipofectamine 3000, following manufacturers instructions. Rnaimax transfection for huvec lipofectamine thermo. However, i could only reach at most 2030% transfection efficiency. Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations. A plasmid expressing the enhanced gfp egfp was used for transfection, followed by flow cytometry of transfected huvec to determine the proportion of egfpexpressing cells as a measure of transfection efficiency. Lipofectamine2000 cd reagent is a proprietary animal originfree formulation for transfecting nucleic acids into eukaryotic cells. Lipofectamine 2000 and lipofectamine ltx invitrogen, carlsbad, ca, usa gave the highest transfection efficiencies of the reagents. We did not find substantial transfection efficiency variations in different batches of lipofectamine 2000.
Transfection of huvec cells were performed by using. Transfection of human endothelial cells cardiovascular. Hmec1, human dermal microvascular endothelial cells, plasmid dna, lipofectamine ltx. Important guidelines for transfection follow these important guidelines when transfecting dna into huvec cells using lipofectamine ltx reagent. Three cell densities of hepg2 cells were transfected with gapd sirna 100 nm using a range of volumes 0. Our study demonstrated that a small selection of commercially available chemical transfection reagents was able to transfer exogenous genes efficiently to primary human cells. This comprehensive guide covers all of the different transfection methods and discusses the best method to use in every sample type. Lipofectamine 2000 and lipofectamine rnaimax transfection. Lipids are a broad class of fatsoluble biomolecules, such as fats, oils, and waxes.
Human umbilical vein endothelial cell huvec avalanche transfection reagent is a new, proprietary solution specifically designed for transfection on huvec. Neuropilin 1 regulates attachment in human endothelial. Human umbilical vein endothelial cell huvec avalanche. Follow these important guidelines when transfecting dna into huvec cells using lipofectamine ltx reagent. Transfection efficiencies were optimal around 40% of transfected cells per spot with 11. Pdf optimizing transfection of primary human umbilical. Altogen cro offers in vivo rnai services, tumor xenograft models, toxicology testing, stable. Invitrogen lipofectamine transfection reagent protocols have been.
Polyplus transfection developped jetpei huvec that is really efficient for huvec cells 70% transfection efficiency. Scaling up or down lipofectamine 3000 reagent transfections use the following table to scale the volumes for your transfection experiment. We are using huvecs to study the mechanism of kinins in inhibitingn tnfainduced expression icam and pselectin by endothelial cells, but we kept. Lipofectamine ltx was identified as the optimal transfection reagent as a result of. Lipofectamine2000 cd reagent complexes can be added directly to cells in culture medium. Arpe19 transfection kit retinal pigment epithelium. Optimized conditions for successful transfection of human. Make sufficient 10% fbs in optimem media wo antibiotics sterile filter 2. 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 addition of antibiotics to media during transfection may result in cell death, and has not been tested for huvec cells. After 4 hours the transfection medium was removed, the cells were washed twice with phosphatebuffered saline pbs, and then maintained in complete huvec medium for 48 to 72 hours. High efficiency transient transfection of huvec was achieved by carefully controlling the conditions of huvec isolation and cultures, the choice. Higher densities can lead to spontaneous differentiation. Conditions were set up according to the manufacturers recommendations both for lipofectamine. Transfecting sirna using lipofectamine rnaimax pin. Transfection reagents are highly efficient for dna and sirna transfection in vivo and in vitro. Invitrogen lipofectamine 2000 transfection reagent is a proprietary formulation for the transfection of nucleic acids dna and rna into eukaryotic cells providing the following advantages. Cellspecific transfection protocols thermo fisher scientific us. Several cell densities and lipid volumes were investigated to determine optimal transfection conditions, shown by the shaded boxes. Apdlriedope for transfection of human endothelial cells reflects the large potential of liposomemediated gene transfer, as systematic structural changes during the last few years led to the discovery of new liposome formulations with advantageous properties for different applications. Lipofectamine 2000 transfection reagent dartmouth college.