Transfection protocol lipofectamine 3000 transfection reagent a549 lung cancer cells complete growth medium component cat. Protocol 6 well dish plate cells and grow to 70% confluence in 2 ml media without antibiotics dmem with 10% fbs, no psg. Lipofectamine 2000 dna transfection reagent protocol. I wonder if somebody has an optimized protocol for nih3t3 transfection using lipofectamine2000, regarding seeding numbers, transfection medium, quantity of dna and lipofectamine, and transfection.
Lipofectamine transient transfection of cos7 cells to analyze. At 72 hours posttransfection the cells were analyzed by western blot for protein expression levels normalized by total protein, 10 g of total protein loaded per each well. Full text an efficient method for in vitro gene delivery via regulation. The trusted, simple, broadspectrum transfection reagent for most cell lines. The proprietary formulation of lipids and polymers ensures the highest possible transfection efficiencies and. How to increase transfection efficiency when lipofectin 2000. Have you even transfected nih3t3 fibroblasts using pei. Lipofectamine transient transfection of cos7 cells to.
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. Researchers use lipofectamine 2000 reagent for sirna and shrnabased gene knockdown experiments, as well as for gene expression studies. Highest transfection efficiency in many cell types and formats e. However, the lipo 2000 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%. Protocol for lipofectamine transfection in nih3t3 cells using cells plated in 8well plates 1 mix 0. Following on from other products in the lipofectamine range, lipofectamine ltx is a lipid based reagent for transfecting dna and vector based rnai into eukaryotic cells. Because of the low infection efficiency, i am a bit worried the same thing will happen on transient transfection using lipofectamine 2000 on nih3t3. Use a range of several ul of lipofectamine and ug dna to increase viability after transfection and efficiency. Nih3t3 transfection kit swiss mouse fibroblasts altogen. Have you even transfected nih3t3 fibroblasts using pei reagent. Nih3t3 standard transfection protocol 24well plate. Nih3t3 cells also known as 3t3 cells are a standard fibroblast cell line utilized in biological research. For additional information on scaling your transfection reaction, see page 4. Metafectene pro, metafectene, fugene, lipofectamine 2000.
However, for cotransfection of sirnachimera and plasmid dna, lipofectamine 2000 should be used. Add 100l of optimum to the eppendorf tube to dilute the dna and mix by tapping. Despite this, a satisfactory mechanismbased explanation of their. Transfection protocol lipofectamine 3000 transfection reagent mdamb231 breast cancer cells complete growth medium component cat. Considerations for optimal transfection using lipofectamine 2000 2. Add 6l of lipofectamine2000 from biostores and 100 l of. Protocol 1 stable transfection using lipofectamine 2000 in 6 well plates. The intracellular trafficking mechanism of lipofectamine. To tranfect nih3t3 cells in 24well plate, follow optimum condition for transfection and transfection procedure. 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. Invitrogen have added a new product, lipofectamine ltx, to their extensive range of transfection products. Methods and materials in one experiment, healthy, low.
Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000. Protocol for lipofectamine transfection in nih3t3 cells. The total volume of the transfection mixes was 100ul, and it was added to the medium already in the wells. Transfection protocol lipofectamine 3000 transfection reagent. Different titration methods of lvv stocks, as well as different culture media, culture durations, cell.
Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. Transfection commonly refers to the introduction of nucleic acids into eukaryotic cells, or more specifically, into animal cells. Before seeding cells for virus package, make sure hek293t cells are in rapid replication state by growth cells to approximately 75%80% confluence in a 10 cm cell culture dish and passaging at 1. Using this protocol, the immortal cell line begins to thrive and stabilize in cell culture. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines.
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. Optimization of lentiviral vector production using. For all transfection of multiple wells in 6 well plate, multiply cdna, optimem, and lipofectamine quantity used by number of wells to transfect. Each reagent was used to transfect hek 293, hela, lncap, a549, and. Dna plasmid expressing gapdh or sirna targeting gapdh were transfected into nih3t3 cells following altogen biosystems transfection protocol. Hela cells, near confluent sixwell plates were transfected with. 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. Metafectene pro, metafectene, fugene, lipofectamine 2000 experimental procedures transfection protocol. 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.
T100100 and plated at high density 300,000 cells 500mlwell in serumfree. Transfection protocol of lipofectamine rnaimax can be obtained from the invitrogen website. Microscopic observations of nih3t3 murine embryonic fibroblasts preadipocytes transfected with viromer red and lipofectamine 3000 anonymous data. Advanced transfection with lipofectamine 2000 reagent. 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. Lipofectamine 2000 lipo2000 is one of the most widely used commercial cationic lipids with high efficiency for in vitro gene transfer. Nih3t3 cell avalanche transfection reagent is a new, proprietary solution specifically designed for transfection on nih3t3 cells. Cut plasmid with an appropriate restriction enzyme to produce a linear fragment for transfection. Lipofectamine 2000 transfection reagent is versatile transfection reagent that has been shown to effectively transfect widest variety of adherent and suspension cell lines. For your convenience, the essential components of this protocol are now available in the gibco lung cancer starter kit. Lipofectamine 2000 reagent protocol 202lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. Nih3t3 cell line transfection, expression and culture.
Transpass p is a protein transfection reagent that can deliver intact functional proteins and peptides with high efficiency into live mammalian cells via endocytosis. Invitrogen lipofectamine transfection reagent protocols have been optimized. Transfecting a human neuroblastoma cell line with monster. Gibco rpmi 1640 with glutamax supplement 61870036 10% gibco fbs a3160401 1. They have a pretty complete pdf document about retroviral gene. Sep 25, 2007 lipofectamine ltx is an improvement on other invitrogen transfection reagents as the protocol has been simplified into a simple single tube method that can be scaled up or down according to the size of culture plate in use. Methods and materials in one experiment, healthy, low passage number, log phase nih3t3 cells were detached from culture dishes using the detachin cell detachment solution amsbio cat. This reference provides a recommended procedure to transfect plasmid dna into nih3t3 mouse embryonic fibroblasts atcc cat. To transfect cells with sirna, follow the protocol as described for dna but.
Cells were transfected with a firefly luciferase encoding plasmid dna 0. Transfection protocol transfection components component cat. Lipofectamine 2000 transfection protocol 10cm plates reagents. Nih3t3 is commonly used for in vitro studies, so it gives me the perception its easy to transfect and infect. Mix optimem with dna in an eppendorf 510 g dna0,8 ml optimem 2. For your convenience, the essential components of this protocol are now available in the gibco breast cancer starter kit. The cells were fixed, dapi stained and photographed 24 hours posttransfection. Invitrogen lipofectamine 3000 transfection reagent l3000008 gibco optimem i reduced serum medium 31985062 thermo scientific nunc 24well cellculture treated multidishes 142475 step tube complexation components amount per well 24well. Prewarm 50ml of optimum stored in cold room at 4c 10min. To perform transfection experiments in other cell culture plates, simply multiply the suggested quantities by the relative surface area of your plate. Hela s3, human cervical carcinoma, sirna, lipofectamine rnaimax.
Transfection of nih3t3 cells, hela, swis 3t3, 293t with. Change the growth medium 4 hours after transfection. Make sure and evenly distribute the cells on the coverslips by swirling plate. To transfect cells with sirna, follow the protocol as. Lipofectamine 2000 transfection for hek293 cells about 6070% transfection rate transfect cells 6well plate. Nih3t3 cell avalanche transfection reagent,will be.
I use 24well plates for transfections in triplicates. For reverse transfection b, a549 cells were plated after adding complexes following the protocol below. Introducing the monster green fluorescent protein phmgfp vector using the transfast transfection reagent and the optimized transfection conditions resulted in the cell notes issue 7 2003 14 neuroscience transfecting a human neuroblastoma cell line with monster green fluorescent protein by natalie betz, ph. Prepare plasmid dnalipid complexes recommend 2 doses of lipid. Make sufficient 10% fbs in optimem media wo antibiotics sterile filter 2. Lipofectamine reagents are widely accepted as goldstandard for the safe delivery of exogenous dna or rna into cells.
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. Lipofectamine transfection of c2c12 cells bridges lab. Nih3t3 cell avalanche transfection reagent,will be provided. Lipofectamine 2000 reagent protocol 202 lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart.
May 11, 2016 lipofectamine reagents are widely accepted as goldstandard for the safe delivery of exogenous dna or rna into cells. How to increase transfection efficiency when lipofectin. Cho k1, hamster ovary, plasmid dna, lipofectamine ltx, 15338100 15338500 pdf. Lipofectamine 2000 protocol, with 2ul of lipofectamine 2000 per well.
Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations. This protocol is for transfecting plasmid dna into mammalian cells. Optimum amount needed is determined from the protocol for lipofectamine 3000 transfection reagent. Cellspecific transfection protocols thermo fisher scientific us. Comparison of transfection efficiency between commercialy. Transfection p19 cells with lipofectamine 2000 the same protocol can be used for 293t cells 1. Plate cells so they will be 7090% confluent at the time of transfection. Lipofectamine ltx reagent is a proprietary, animalorigin free formulation for the transfection of dna into eukaryotic cells with low cytotoxicity. In standard pei and lipo2000 transfection cases, the growth medium. Invitrogen lipofectamine 2000 transfection reagent. Invitrogen lipofectamine 2000 transfection reagent is a versatile transfection reagent that has been shown to effectively transfect a variety of payloads into the widest variety of adherent. Gibco dmem with glutamax supplement a3635201 10% gibco fbs a3160401 proper culture techniques and procedures are an essential part of ensuring successful transfection.
In addition metafectene pro was also used at 1 g plasmid and 4 l reagent 1. A sample protocol is listed here for transfection experiments performed in 6well plates. Assay measure protein expression introduction this protocol shows optimum transfection condition using hilymax in nih3t3 cells. Mix optimem and lipofectamine in another eppendorf 32 l lipofectamine in 0,8 ml optimem 3.
Introducing the monster green fluorescent protein phmgfp vector using the transfast transfection reagent and the optimized transfection conditions resulted in the cell notes issue 7 2003 14 neuroscience transfecting a human neuroblastoma cell line with monster green fluorescent protein by. The aim of the present study was to optimize the polyethylenimine peimediated transfection method in order to simplify the efficient production of lentiviral vectors lvvs, and to compare the capo 4 and peimediated transfection methods for producing lvvs. Lipofectamine ltx transfection reagent from invitrogen. Nanomagnetic gene transfection for nonviral gene delivery. Genscript recommends using lipofectamine 2000 for all transfections. Transfection of fluorescently labeled oligos with lipofectamine 2000 back to table of content back to protocol and application notes.
Maintain p19 cells undifferentiated in mem with 10% serum 7. The proprietary formulation of lipids and polymers ensures the highest possible transfection efficiencies and viabilities for nih3t3 cells. A highly efficient method of transfecting nih3t3 cells. Hi, i transfect nih 3t3 often initially i also used lipofectamine 2000 and had low efficiency, but then switched to lipofectamine ltx slightly more expensive and it works much better.
1289 722 479 1486 969 1593 1023 1427 527 310 630 335 605 1123 1197 1559 1060 734 294 951 1240 99 1508 244 563 1195 328 274 1404 534 792 903 808 874 459 164 447 1228 796 628 723 763 1191