3t3 transfection lipofectamine 2000 pdf

Transfection amounts component 96well 24well 6well dna per well 100 ng 500 ng 2500 ng p3000 reagent per well 0. Transitx2 transfection reagent a dynamic transfection system for the. One thing you should know about 3t3l1 is that they are pretty, pretty hard to transfect. Viafect transfection reagent transfection assay e4981. Lipofectamine 2000 dna transfection reagent protocol. Lipofectamine 3000 reagentefficient, reproducible transfection for biologically relevant cell models introduction. This reagent delivers superior transfection efficiency and improved cell viability for the widest range of hardtotransfect and common cells e. One day before transfection, plate 3 x 10 4 3t3 l1 cells in 400. 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. Metafectene pro, metafectene, fugene, lipofectamine 2000. Several cell densities and lipid volumes were investigated to determine optimal transfection conditions, shown by the shaded boxes. Metafectene pro, metafectene, fugene, lipofectamine 2000 experimental. A sample protocol is listed here for transfection experiments performed in 6well plates.

Nih3t3 cell line transfection, expression and culture. In contrast to the efficiency of gene suppression in hela cells, treatment with lipofectamine 2000 and 50 nm sirna in imcd, 3t3l, pc12, and jurkat cells effected no significant decrease in gapdh protein levels fig. Nih 3t3 mouse embryonic fibroblast cells were initiated from a cell line isolated in 1962 at the new york university school of medicine department of pathology. Bgalactosidase expression was measured 48 hours posttransfection. Transitx2 for crispr plasmid delivery protocol pdf. 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. Add 6l of lipofectamine2000 from biostores and 100 l of. Have somebody done the transfection in 3t3 l1 cell lines. Protocol for lipofectamine transfection in nih 3t3 cells using cells plated in 8well plates 1 mix 0. We recently tested transitx2tm dynamic delivery system headtohead against lipofectamine 2000 for dna transfection of nih3t3 fibroblasts and the. Even adenovirus, at an moi of 2000, lend about 50% of positively infected cells.

Nanomagnetic gene transfection for nonviral gene delivery. Cellspecific transfection protocols thermo fisher scientific us. In the following 48 h, viruses were released into the droplets in each microwell. Edwin li, assistant professor saint josephs university. Add 100l of optimum to the eppendorf tube to dilute the dna and mix by tapping. Comparison of transfection efficiency between commercialy. This new reagent is able to deliver plasmid dna into the fibroblast cell line, nih 3t3, with three fold greater efficiency than the popular commercial transfection reagent, lipofectamine 2000 lf2k. For 25006500 transfections in 60 mm dishes or 500010,000 transfections in 6well plates. Protocol for lipofectamine transfection in nih3t3 cells transfection of mcf7 with lipofectamine 2000 maartje vogel. However, the lipo2000 transfection efficiency is low about 5% to 15% acting on the cell line 3t3 or neuronal cell line gt17 compared to the cell line 293at about 80% to 90%. The optimal volume of lipofectamine and quantity of plasmid. The objective of this work was to examine the potential of oscillating nanomagnetic gene transfection systems magnefectnano for improving the transfection efficiency of nih3t3 mouse embryonic fibroblasts mefs in comparison to other nonviral transfection techniquesstatic magnetofection and the cationic lipid agent, lipofectamine 2000.

To transfect cells with sirna, follow the protocol as described for dna but. Cho k1, hamster ovary, plasmid dna, lipofectamine ltx, 15338100 15338500 pdf. For each transfection sample, prepare stealth rnai lipofectamine 2000 complexes as follows. Altogen cro offers in vivo rnai services, tumor xenograft models, toxicology testing, stable cell line generation, and. Transfection protocol lipofectamine 3000 transfection. To transfect cells with sirna, follow the protocol as. After overnight culture, cells are transfected with specific plasmid or sirna with lipofectamine 2000 according to the manufacturers instruction. Cells should be 6070% confluent at the time of transfection.

Lipofectamine 2000 transfection reagent dartmouth college. Transfection protocol of lipofectamine rnaimax can be obtained from the invitrogen website. If i can give you an advice, i would tell you to go for a lentivirusretrovirus mediated transduction. Highest transfection efficiency in many cell types and formats e. Prepare plasmid dnalipid complexes recommend 2 doses of lipid. Higher densities can lead to spontaneous differentiation. L in cotransfection of lipofectamine 2000 in serumfree media and incubated at 37. Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000. Lipofectamine 2000 right panel around 5% efficiency. No other transfection reagents can match the efficiency, convenience. Using this protocol, the immortal cell line begins to thrive and stabilize in cell culture. Genscript recommends using lipofectamine 2000 for all transfections.

Lipofectamine transfection reagent is no exception as a trusted reagent for a wide range of cell lines. Lipofectamine 3000 reagent is designed to efficiently transfect difficulttotransfect cells, yielding superior transfection performance across the broadest array of cell types. Invitrogen lipofectamine 3000 transfection reagent 0. The proprietary formulation of lipids and polymers ensures the highest possible transfection efficiencies and viabilities for nih3t3 cells. Cellspecific transfection protocols thermo fisher scientific uk. I get about 100% positively transduced cells that way. Lipofectamine 2000 reagent thermo fisher scientific. Transfection of nih3t3 cells, hela, swis 3t3, 293t with. Lipofectamine 2000 volumes on cell transfection efficiency was evaluated. Transfection was measured by luciferase activity using a conventional assay. Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. With proprietary shaved cell transfection protocol, polyjet left panel gives up to 70% gfp positive cells vs. Three cell densities of hepg2 cells were transfected with gapd sirna 100 nm using a range of volumes 0.

Lipofectamine 2000 reagents 3t3 4 4t1 2 a431 2 a549 3 achn 2 bend. Polyfect transfection reagent is based on activated dendrimer technology optimized for the transfection of dna into cos7, nih3t3, hela, 293, and cho cells. Transitx2 dynamic delivery system and lipofectamine 2000 transfection reagent were used to. We recommend the dendrimer transfection reagent, polyfect qiagen which has been optimized for use on nih3t3 cells. Invitrogen lipofectamine 3000 transfection reagent. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 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.

How to increase transfection efficiency when lipofectin. However, for cotransfection of sirnachimera and plasmid dna, lipofectamine 2000 should be used. Nih3t3 cell avalanche transfection reagent,will be. A comparison showing transfection efficiency of polyjet reagent vs. Invitrogen lipofectamine transfection reagent protocols have been. I want to transfect my protein of interest in 3t3 cells. Pdf optimization of sw480 colon cancer cells transfection with. Classical transfection of 3t3l1 adipocytes with sirna. Transfection protocol lipofectamine 3000 transfection reagent skbr3 breast cancer cells complete growth medium component cat. Lipofectamine 2000, and lipofectamine 2000 cd x broad spectrum dna deliveryachieve high expression in many cell types, including hardtotransfect cells and insect cells x animal origin freetransit2020 provides high performance with maximum compatibility description transit2020 reagent is a new and versatile transfection solution for. Nih 3t3, mouse embryonic fibroblast, sirna, lipofectamine rnaimax, 778. Transfection p19 cells with lipofectamine 2000 the same protocol can be used for 293t cells 1. Scaling up or down lipofectamine 3000 reagent transfections. Lipofectamine 2000 transfection for hek293 cells about 6070% transfection rate transfect cells 6well plate.

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. Maintain p19 cells undifferentiated in mem with 10% serum 7. A highly efficient method of transfecting nih3t3 cells. Lipofectamine 2000 invitrogen according to the manufacturers protocol. Fibroblast 3t3, cos7 myoblast c2c12, l6 crl1458 hepatocyte hepg2, huh7 erythroleukemic cell k562.

Plate cells so they will be 7090% confluent at the time of transfection. Transformation of human liver lo2 cells mediated by. 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. Lipofectamine ltx reagent is a proprietary, animalorigin free formulation for the transfection of dna into eukaryotic cells with low cytotoxicity. Does anyone has any experience in transfecting this cell line. I use 24well plates for transfections in triplicates. This reference provides a recommended procedure to transfect plasmid dna into nih3t3 mouse embryonic fibroblasts atcc cat. Nih3t3 cells also known as 3t3 cells are a standard fibroblast cell line utilized in biological research. Protocol for lipofectamine transfection in nih3t3 cells. Transfection reagents are highly efficient for dna and sirna transfection in vivo and in vitro. Nih3t3 cell avalanche transfection reagent is a new, proprietary solution specifically designed for transfection on nih3t3 cells. Sntb2 is a cellular adaptor protein affecting several receptors. To perform transfection experiments in other cell culture plates, simply multiply the suggested quantities by the relative surface area of your plate.

Altogen biosystems provides in vivo transfection reagents, over 100 preoptimized in vitro transfection kits for cell lines and primary cells, and electroporation delivery products. Gibco dmem with glutamax supplement 10566016 10% gibco fbs a3160401 0. Lipofectamine 2000 reagent protocol 202 lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. Prewarm 50ml of optimum stored in cold room at 4c 10min. Mammalian cell penetration, sirna transfection, and dna. Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations.