Few cells were transfected by lipofectamine plus but it was not too. Xtremegene sirna transfection reagent efficiently delivers short interfering rna sirna into many commonly used cell types including hela, nih 3t3, hek293, chok1, and cos7, and several hardto. Viafect reagent offers performance similar to or better than that of lipofectamine 2000 reagent, and it may be suitable for some cell lines where other lipidbased reagents result in lower transfection efficiencies table 1. Transfection protocol lipofectamine 3000 transfection reagent mcf10a breast cancer cells complete growth medium component cat. Transfecting stealth rnai or sirna into 3t3 l1 cells. Lipofectamine 3000 reagent thermo fisher scientific kr. Achieve superior transfection efficiency while maintaining low toxicity lipofectamine 3000 reagent maintains a high transfection efficiency within a robust dynamic range of lipid doses for quick and easy optimization. Transfection of nih3t3 cells, hela, swis 3t3, 293t with. Lipofectamine transfection of c2c12 cells bridges lab. Protocol 1 stable transfection using lipofectamine 2000 in 6 well plates. Transfection protocol lipofectamine 3000 transfection. Lipofectamine 2000 transfection for hek293 cells about 6070% transfection rate transfect cells 6well plate. Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations.
Gibco hams f12 nutrient mix 11765054 5% horse serum. For additional information on scaling your transfection reaction, see page 4. Protocol for lipofectamine transfection in nih 3t3 cells using cells plated in 8well plates. This reference provides a recommended procedure to. As always, use good cell culture practices and wear your. Hek 293 cells were transfected with pgenesil1 using lipofectamine 2000. Lipofectamine 2000 had highest efficiency but also highest toxic effect. Lipofectamine transfection reagent is no exception as a trusted reagent for a wide range of cell lines. Follow these important guidelines when transfecting stealth rna or sirna into 3t3 l1 cells using lipofectamine 2000. A sample protocol is listed here for transfection experiments performed in 6well plates. Viafect reagent offers performance similar to or better than that of lipofectamine 2000 reagent, and it may be suitable for. Lipofectamine 2000 reagent protocol 202lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart.
Does anyone has any experience in transfecting this cell line. Lipofectamine 3000 transfection reagent leverages our most advanced lipid nanoparticle technology to provide superior transfection performance with improved. Xtremegene sirna transfection reagent polymer reagent. Transfection protocol of lipofectamine rnaimax can be obtained from the invitrogen website. This reference provides a recommended procedure to transfect plasmid dna into nih3t3 mouse embryonic fibroblasts atcc cat. How to increase transfection efficiency when lipofectin. Lipofectamine or lipofectamine 2000 is a common transfection reagent, produced and sold by invitrogen, used in molecular and cellular biology. Table 2 examines transfection efficiency and cell viability under optimal conditions for each reagent 24 hours after transfection. Transit3t3 transfection kit transit3t3 reagent and 3t3 authority reagent. On the day of transfection, which should be 1 day following cell plating, perform the following steps, which have been optimized for a single well of a 24well plate using lipofectamine 3000 transfection reagent. Transfecting plasmid dna into nih3t3 cells using lipofectamine.
Choosing the right transfection reagent for optimal efficiency. The cells were fixed and photographed 24 hours post transfection. Nih3t3 cell avalanche transfection reagent,will be provided. Lipofectamine 3000 transfection reagent leverages our most advanced lipid nanoparticle. Transfection protocol lipofectamine 3000 transfection reagent. Add 100l of optimum to the eppendorf tube to dilute the dna and mix by tapping. Xgal staining of nih3t3 cells 24 hours after transfection by fugenehd, fugene6 and competitor lipofectamine 2000, 5,000 cellswell in 96 well plate click here to view the protocol fugenehd. While lipofectamine 2000 reagent demonstrated transfection efficiencies only slightly lower those of viafect reagent, it was considerably more toxic to hela cells at all tested ratios of reagent to dna. While lipofectamine 2000 reagent demonstrated transfection efficiencies only slightly lower. Lipofectamine 3000 reagent is designed to efficiently transfect difficulttotransfect cells, yielding superior transfection performance across the broadest array of cell types. How i can get good transfection efficiency in these cells.
Lipofectamine 2000 transfection reagent from invitrogen. Transfection guide overview of transfection methods. Lipofectamine 3000 reagent provided higher gfp transfection efficiency than lipofectamine 2000 and fugene hd reagents for all five cell lines. Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. 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. One thing you should know about 3t3 l1 is that they are pretty, pretty hard to transfect. Lipofectamine 2000 reagent thermo fisher scientific. 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 delivery reagents are limited. Optimized conditions for successful transfection of human. Effi ciency and ofp expression were analyzed 72 hours posttransfection and a u2os and b hepg2 cells showed 4fold and 80fold improvement with lipofectamine. Comparative transfection efficiency of jetprime versus its main competitor. 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. I want to transfect my protein of interest in 3t3 cells. If i can give you an advice, i would tell you to go for a lentivirusretrovirus mediated transduction.
Achieve superior transfection efficiency while maintaining low toxicity lipofectamine 3000 reagent maintains a high. Lipofectamine 2000 is a cationic liposome based reagent that provides high transfection efficiency and high levels of transgene expression in a range of mammalian cell types in vitro using a. The cells were photographed 24 hours posttransfection. Mouse neuronal cells were transfected with a gfpexpressing plasmid using either primefect transfection reagent c, or lipofectamine 2000 d. 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. How to increase transfection efficiency when lipofectin 2000. To perform transfection experiments in other cell culture plates, simply multiply the suggested quantities by the relative surface area of your plate. How to increase transfection efficiency when lipofectin 2000 was used on the cell line 3t3 or neuronal cell line gt17. Nih3t3 cells were transfected with a luciferase expression plasmid using the transit3t3 transfection kit or the transitx2 dynamic delivery system at the reagenttodna ratios indicated beneath each bar.
Conditions were used according to the manufacturers recommendation for both lipofectamine 2000 and for jetprime. It is used to increase the transfection efficiency of rna. 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. Lipofectamine 2000 transfection reagent is versatile transfection reagent that has been shown to effectively transfect widest variety of adherent and suspension cell lines. Vero hepg2 nih 3t3 lipofectamine 2000 cd is a 100% synthetic version of.
The transfection protocol for lipofectamine 3000 was developed to be easy to use while. Transfecting stealth rnai or sirna into 3t3 l1 cells using. Lipofectamine 2000 dna transfection reagent protocol. The 3t3 cell line was originally established from a swiss albino mouse embryo tissue in 1962 by two scientists at new york university school of medicine. All products were used according to manufacturers protocol. However, the lipo2000 transfection efficiency is low about 5% to 15% acting on. Transfections were performed in 96well plates using 0. I get about 100% positively transduced cells that way. Even adenovirus, at an moi of 2000, lend about 50% of positively infected cells. Lipofectamine 2000 transfection reagent dartmouth college. Transfection efficiencies lower than 50% were not reported in this table. Jul 24, 2007 lipofectamine 2000 is a cationic liposome based reagent that provides high transfection efficiency and high levels of transgene expression in a range of mammalian cell types in vitro using a simple protocol. Transfection in 3t3 l1 is definitely hard compared to other cell lines. One thing you should know about 3t3l1 is that they are pretty, pretty hard to transfect.
Transfection protocol of lipofectamine rnaimax can be. The following reagent to dna ratios were tested according to the manufacturers instructions. Transient plasmid dna transfection into hela and hacat cells. Genscript recommends using lipofectamine 2000 for all transfections. Invitrogen lipofectamine 2000 transfection reagent. Invitrogen lipofectamine 3000 transfection reagent fisher. The proprietary formulation of lipids and polymers ensures the highest. Nih3t3 cells also known as 3t3 cells are a standard fibroblast cell line utilized in biological research. Fugene hd, jetpei, lipofectamine 2000, and superfect for gene transfection. Prewarm 50ml of optimum stored in cold room at 4c 10min.
As always, use good cell culture practices and wear your personal protective. Invitrogen lipofectamine 3000 transfection reagent. Nih3t3 cell avalanche transfection reagent,will be. I used lipofectamine 2000, 3000, pei, fugene 6 and hd, electroporation and. I used lipofectamine 2000 to transfect preadipocytes for 24 hours and then differentiated the cells for 6 days. Lipofectamine 3000 reagent outperforms invitrogen lipofectamine 2000 and fugene hd reagents. Does someone have successful transfection in 3t3l1 cells.
Invitrogen lipofectamine 3000 transfection reagent 0. Transitx2 exhibits superior gene expression in nih3t3 cells. Protocol 6 well dish plate cells and grow to 70% confluence in 2 ml media without antibiotics dmem with 10% fbs. Transit3t3 transfection kit research reagent products. The proprietary formulation of lipids and polymers ensures the highest possible transfection efficiencies and viabilities for nih3t3 cells. Transfection efficiency was assessed by facs analysis in various cell lines 24 h after transfection in 24well plates. Lipofectamine 2000 transfection reagent is a proprietary formulation for the transfection of nucleic acids dna and rna. Ive attempted to transfect nih3t3 cells using lipofectamine 3000 and ive. Transient plasmid dna transfection into hela and hacat. Lipofectamine 3000 reagent maintains a high transfection efficiency within a robust dynamic range of lipid doses for quick and easy optimization. The lipofectamine 3000 is the bees knees though, i managed to get loads of transfects from.
Transfecting plasmid dna into nih3t3 cells using lipofectamine ltx reagent. Lipofectamine ltx reagent is a proprietary, animalorigin free formulation for the transfection of dna into eukaryotic cells with low cytotoxicity. To perform transfection experiments in other cell culture plates, simply multiply the. 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 lipofectamine 3000 transfection reagent l3000008. Lipofectamine 2000 invitrogen according to the manufacturers protocol. Lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. The use of these different lipofectamine 2000 amounts for transfection showed no significant differences. I have performed lipofectamine transfection of cdna3. Transpass p is a protein transfection reagent that can deliver intact functional proteins and peptides with high efficiency into live mammalian cells via endocytosis.
Optimemin ul, equal volume for dna and lipofectamine lipofectaminein ul 25. How to increase transfection efficiency when lipofectin 2000 was. On the day of transfection, which should be 1 day following cell plating, perform the following steps, which have been optimized for a single well of a 24well plate using lipofectamine 3000 transfection. Nih3t3 cell avalanche transfection reagent is a new, proprietary solution specifically designed for transfection on nih3t3 cells. Transfection guide overview of transfection methods promega. Comparison of primefect tm and lipofectamine 2000 in plasmid transfection on mouse neuronal cells. No other transfection reagents can match the efficiency, convenience, and gentleness of nih3t3 cell avalanche transfection reagent for transfection on nih3t3 cells. Protocol for lipofectamine transfection in nih3t3 cells. Lipofectamine 3000 reagent thermo fisher scientific us.
When used in combination with plus reagent, lipofectamine transfection reagent has been shown to work well with cells such as bhk21, nih 3t3, cos1, fibroblasts, keratinocytes, ht29, mrc5, and skbr3. Comparison of transfection efficiency of nonviral gene. Highest transfection efficiency in many cell types and formats e. Add 6l of lipofectamine2000 from biostores and 100 l of. Theres a difference between the two protocols lipofectamine 3000 has an extra reagent that needs to be added. Invitrogen lipofectamine 2000 transfection reagent fisher. Researchers use lipofectamine 2000 reagent for sirna and shrnabased gene knockdown experiments, as well as for gene expression studies. This protocol is for transfecting plasmid dna into mammalian cells. Lipofectamine 2000 reagent is a lipidderived reagent designed to deliver exogenic nucleic acids dna andor rna into eukaryotic cells, either adherent or in suspension. Here, we report the results of a transfection trial with three promega reagents and another widely used. Xtremegene hp dna transfection reagent is a proprietary blend of lipids and other components supplied in 80% ethanol, filtered through 0. Comparison of transfection efficiency of nonviral gene transfer reagents. To transfect cells with sirna, follow the protocol as described for dna but. However, for cotransfection of sirnachimera and plasmid dna, lipofectamine 2000 should be used.
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