Invitrogen lipofectamine 2000 transfection reagent 1. Make sufficient 10% fbs in optimem media wo antibiotics sterile filter 2. 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. Transfecting plasmid dna into nih3t3 cells using lipofectamine. Cells in a 6 well dish, plated and at 70% confluence. To transfect cells with sirna, follow the protocol as described for dna but. According to the manufacturers instructions, cells were grown overnight. Advanced transfection with lipofectamine 2000 reagent. 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. Thee two most important factors in choosing transfecting reagent are. Invitrogen lipofectamine 2000 transfection reagent. Protocol for lipofectamine transfection in nih3t3 cells using cells plated in 8well plates 1 mix 0. If i were to try, i would start with calcium phosphate much cheaper and if this doesnot work, i would try fugene or lipofectamine.
Prior to mixing with dna, lipofectamine 2000 should be diluted in optimem i. 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. Transitx2 dynamic delivery system outperforms lipofectamine reagents. Invitrogen lipofectamine 2000 transfection reagent life. Feb 18, 2016 lipofectamine crisprmax is more robust than lipofectamine rnaimax and several other transfection reagents in delivery of cas9 proteingrna complexes into a variety of 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. Depending on the size of culture vessel being used, the lipofectamine 2000 is diluted between 25 and 50fold. For nih3t3, nanomagnetic transfection magnefectnano resulted in 22% transfected cells.
Nih3t3 standard transfection protocol 24well plate. Nih3t3 transfection kit swiss mouse fibroblasts altogen. Application note gene delivery solutions stem cell. Nih3t3 cells also known as 3t3 cells are a standard fibroblast cell line utilized in biological research. Two microliters of lipofectamine was diluted in 25. Because of the low infection efficiency, i am a bit worried the same thing will happen on transient transfection using lipofectamine 2000 on nih3t3. Lipofectamine ltx transfection reagent from invitrogen. Mar 27, 2007 lipofectamine 2000 reagent is a lipidderived reagent designed to deliver exogenic nucleic acids dna andor rna into eukaryotic cells, either adherent or in suspension. Xtremegene 9 dna transfection reagent is a nonliposomal multicomponent reagent for. Theres a difference between the two protocols lipofectamine 3000 has an extra reagent that needs to be added. Transfecting a human neuroblastoma cell line with monster. Xtremegene 9 dna transfection reagent polymer reagent.
When using the other vessel, refer to table 2 and adjust the amounts of cells, medium, dna and hilymax in proportion to the relative surface area. Scaling up or down lipofectamine 3000 reagent transfections use the following table to scale the volumes for your transfection experiment. 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. For nih 3t3 cells, near confluent sixwells plates were transfected with 1. Following staining, the spectramax minimax 300 imaging cytometer was used to count cells using either the nuclear red staining red curve or stainfree cell detectiontechnology blue curve as the basis for cell identification. Jul 17, 2007 lipofectamine 2000 is a lipid based transfection reagent for eukaryotic cells. Gibco rpmi 1640 medium with glutamax supplement 61870036 10% gibco fbs a3160401 25 mm gibco hepes 1 m 15630080 1. Transfection p19 cells with lipofectamine 2000 the same protocol can be used for 293t cells 1. The reagent provides high transfection efficiency in many cells types and formats.
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. Nih3t3 cell line transfection, expression and culture. Stem cell transfection guide introduction stem cells continue to show immense promise for the future of regenerative medicine and personalized therapeutic treatments. This protocol shows optimum transfection condition using hilymax in nih3t3 cells. For your convenience, the essential components of this protocol are now available in the gibco liver cancer starter kit. Lipofectamine 3000 reagent thermo fisher scientific. Transfection protocol lipofectamine 3000 transfection reagent mcf10a breast cancer cells complete growth medium component cat.
Mar 03, 2014 to determine the required amount of lipofectamine 2000 for forming of lipoplexes, different amounts of lipofectamine 2000, 1, 2, 4, 6. Ive tried calcium phospahte and electroporation but no luck. Lipofectamine 2000 reagent protocol 202lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. It is used to increase the transfection efficiency of rna including mrna and sirna or plasmid dna into in vitro cell cultures by lipofection. Xgal staining of nih3t3 24 hours after transfection fugene hd. Choosing the right transfection reagent for optimal efficiency. Lipofectamine 2000 cd reagent is a proprietary animal originfree formulation for transfecting nucleic acids into eukaryotic cells. Native promoters are more problematic, but viral promoters should express in most standard transfections. Comparison of transfection efficiency between commercialy. How to increase transfection efficiency when lipofectin 2000 was.
Invitrogen transfection reagent, recommended transfection sku, protocol. Add 100l of optimum to the eppendorf tube to dilute the dna and mix by tapping. Lipofectamine 2000 transfection reagent from invitrogen. Xtremegene 9 dna transfection reagent is a nonliposomal multicomponent reagent for experiments involving cellular analysis. Gibco hams f12 nutrient mix 11765054 5% horse serum 1 mm gibco sodium pyruvate 160070 25 mm gibco hepes 15630106 proper culture techniques and procedures are an essential. Nih3t3 are derived from a cell line of a swiss mouse embryo established in 1962 by two scientists at the department of pathology in the new york university school of medicine. Optimum amount needed is determined from the protocol for lipofectamine 3000 transfection reagent. Agree, lipofectamine is a huge overkill for 3t3 cells. Add 6l of lipofectamine2000 from biostores and 100 l of. Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations. Amounts of dna and reagent jetprime and competitors per well in 6well plate for transfection according to manufacturers recommendations. Lipofectamine or lipofectamine 2000 is a common transfection reagent, produced and sold by invitrogen, used in molecular and cellular biology. Nih3t3 is commonly used for in vitro studies, so it gives me the perception its easy to transfect and infect.
Lipofectamine 2000 reagent protocol 202 lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. Invitrogen lipofectamine transfection reagent protocols have been optimized. Increase or decrease the amount of dissociation medium needed proportionally for culture vessels of other sizes. I have tried many different transfection reagents for nih3t3. Higher densities can lead to spontaneous differentiation. Researchers use lipofectamine 2000 reagent for sirna and shrnabased gene knockdown experiments, as well as for gene expression studies. Lipofectamine 2000 transfection protocol 10cm plates reagents. Increasing the posttransfection period increases toxicity. Lipofectamine ltx reagent is a proprietary, animalorigin free formulation for the transfection of dna into eukaryotic cells with low cytotoxicity. For additional information on scaling your transfection reaction, see page 4. Refer to the cell lines database for a list of cell types successfully. Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000. Highest transfection efficiency in many cell types and formats e. Mouse embryonic fibroblasts nih3t3 is a type of murine embryonic fibroblast cells mef.
Fluorescent protein exhibited persistent expression in nih3t3 cells over a 7day period, again suggesting the nontoxic nature. The 3t3 cell line has become the standard fibroblast cell line. The cells were fixed, dapi stained and photographed 24 hours posttransfection. A549 a or mdck b cells were transfected with luciferase encoding plasmid dna using either transitx2 mirus bio, lipofectamine 2000 thermo fisher scientific or lipofectamine 3000 thermo fisher scientific for 24 hours at indicated reagenttodna ratios or reagenttop3000todna ratio. Transfecting a human neuroblastoma cell line with monster green fluorescent protein. Invitrogen have added a new product, lipofectamine ltx, to their extensive range of transfection products.
Optimized conditions for successful transfection of human. Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. Prewarm 50ml of optimum stored in cold room at 4c 10min. How to increase transfection efficiency when lipofectin 2000. We have had no problems with native promoters and viral promoters. Few cells were transfected by lipofectamine plus but it was not. Cells were transfected with a firefly luciferase encoding plasmid dna 0. Cellspecific transfection protocols thermo fisher scientific us. Nih 3t3, mouse embryonic fibroblast, plasmid dna, lipofectamine 3000.
This reference provides a recommended procedure to transfect plasmid dna into nih3t3 mouse embryonic fibroblasts atcc cat. The best results were obtained with lipofectamine 3000, but my protein of interest is downregulated even with the empty plasmid. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. Luciferase expression was compared at 24 hours posttransfection using a standard luciferase assay. Using this protocol, the immortal cell line begins to thrive and stabilize in cell culture. Maintain p19 cells undifferentiated in mem with 10% serum 7. While lipofectamine 2000 reagent demonstrated transfection efficiencies only slightly lower those of viafect. Chok1, cos7, hela, hepg2, huh7, huvec, imr90, mcf7, mef, and nih3t3. Hd and lipofectamine 2000, respectively, in laboratorymade mef cells. The cells were fixed and stained with a nuclear dye. However, delivery of the cas9 rnps by neon electroporation was effective across all different cell lines tested and often was the only method that could produce indels. 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. Nih3t3 cell avalanche transfection reagent,will be. Transfection of nih3t3 cells, hela, swis 3t3, 293t with.
Scaling up or down lipofectamine 3000 reagent transfections. 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. Has anyone tried using lipofectamine 3000 to transfect nih. I used home made calcium phosphate with no problems. Invitrogen lipofectamine 2000 transfection reagent 15ml. Lipofectamine 2000 reagent is a lipidderived reagent designed to deliver exogenic nucleic acids dna andor rna into eukaryotic cells, either adherent or in suspension. Lipofectamine 2000 transfection reagent dartmouth college. High throughput test of transfection efficiency determined as rlumg on nih3t3 cells after transfection of luciferase reporter gene by using our 172 proprietary transfection formulas and several most popular commercial transfection reagents. Transpass p is a protein transfection reagent that can deliver intact functional proteins and peptides with high efficiency into live mammalian cells via endocytosis. Comparative analysis of nonviral transfection methods in mouse.
Protocol for lipofectamine transfection in nih3t3 cells. Lipofectamine 2000 reagent thermo fisher scientific. Lipofectamine 2000 and lipofectamine rnaimax transfection. Transfection protocol lipofectamine 3000 transfection reagent gct lung cancer cells complete growth medium component cat. Product is guaranteed for 6 months from the date of shipment if. Lipofectamine transfection of c2c12 cells bridges lab protocols. I use 24well plates for transfections in triplicates. 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. To tranfect nih3t3 cells in 24well plate, follow optimum condition for transfection and transfection procedure. Hi, i transfect nih 3t3 often initially i also used lipofectamine 2000 and had low. 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. Improved delivery of cas9 proteingrna complexes using. Xtremegene 9 dna transfection reagent is a proprietary blend of lipids and other components supplied in 80% ethanol, filtered through 0. 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.
Are you sure your promotersterminators are set up properly. How to increase transfection efficiency when lipofectin. Have somebody done the transfection in 3t3 l1 cell lines. Transfection protocol lipofectamine 3000 transfection. A highly efficient method of transfecting nih3t3 cells. It can be used for transfection of adherent or suspension cells with dna or rna including stealth rnai and sirna and can be used for either transient transfections or to create stable cell lines. 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. Visit technical service for other specialized transfection protocols including cell type specific advice on use of plus reagent and antibiotics, and a protocol for.
Importantly, this improved efficiency does not come at the expense of cell viability since cells transfected using geneporter 3000 reagent are significantly healthier and exhibit higher viability than cells transfected using lf2k. Lipofectamine 2000 cd reagent complexes can be added directly to cells in culture medium. Since the discovery of induced pluripotent stem cells ipscs in 2006, researchers have. The fugene hd protocol database provides optimization guidelines for a variety of cell lines. Effi ciency and ofp expression were analyzed 72 hours posttransfection and a u2os and b hepg2 cells showed 4fold and 80fold improvement with lipofectamine.
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