Three cell densities of hepg2 cells were transfected with gapd sirna 100 nm using a range of volumes 0. Lipofectamine is formulated to give high transfection efficiency with little toxicity to cells. I am about to start my stable transfections with lipofectamine 2000. I have never tried lipo 3000, but with lipo 2000 we often experienced toxicity.
Cut plasmid with an appropriate restriction enzyme to produce a linear fragment for transfection. Viafect reagent offers performance similar to or better than that of lipofectamine 2000 reagent, and it may be suitable for some cell. Here, we report the results of a transfection trial with three promega reagents and another widely used transfection reagent. Lipofectamine transfection of c2c12 cells bridges lab. Invitrogen claims that this reagent gives the highest protein expression levels in the widest variety of adherent and suspension. How does transfection with lipofectamine 2000 work. Gibco rpmi 1640 medium, with glutamax supplement 61870036 10% gibco fbs a3160401 1 mm gibco sodium pyruvate 160070 proper culture techniques and procedures are an essential part of ensuring successful.
Transfex transfection of plasmid dna into lncap fgc cells lncap fgc atcc cat. The reagent provides high transfection efficiency in many cells types and formats. Lipofectamine 2000 cd reagent is a proprietary animal originfree formulation for transfecting nucleic acids into eukaryotic cells. Altogen cro offers in vivo rnai services, tumor xenograft models, toxicology testing, stable cell line generation, and. Transfection the delivery of dna or rna into eukaryotic cells is a powerful tool used to study and control gene expression. Lipofectamine 3000 reagent outperforms invitrogen lipofectamine 2000 and fugene hd reagents.
I have used lipofectamine2000 and superfect as well it does not help, efficiency is very low. Highest transfection efficiency in many cell types and formats e. Invitrogen lipofectamine 3000 transfection reagent. To test the transfection efficiency of the reverse transfection approach, a luciferaseencoding plasmid was reverse transfected into hek293, hep g2 and jurkat cells using fugene 6, fugene hd, transitlt1 and lipofectamine 2000 transfection reagents.
Lipofectamine or lipofectamine 2000 is a common transfection reagent, produced and sold by invitrogen, used in molecular and cellular biology. Transfection efficiency was assessed by facs analysis in various cell lines 24 h after transfection in 24well plates. Cells were transfected with a firefly luciferase encoding plasmid dna 0. Transfex transfection of plasmid dna into lncap fgc. Hela, lncap, hepg2, and a549 cell lines in a 96well format, and green fluorescent protein. Altogen biosystems provides in vivo transfection reagents, over 100 preoptimized in vitro transfection kits for cell lines and primary cells, and electroporation delivery products. Transfection was measured by luciferase activity using a conventional assay. Transfex transfection of plasmid dna into lncap fgc cells. I want to try to use hela cell and lipofectamine 2000 to. The vector contained an ofp reporter gene and was transfected. I am trying to optimize the transfection by using lipofectamine 2000 for 2000 ng of plasmid dna well with different amounts of lipofectamine 2000 5ul, 10ul in the 6well plate.
It is used to increase the transfection efficiency of rna including mrna and sirna or plasmid dna into in vitro cell cultures by lipofection. Theres a difference between the two protocols lipofectamine 3000 has an extra reagent that needs to be added. Lipofectamine 2000 transfection reagent is versatile transfection reagent that has been shown to effectively transfect widest variety of adherent and suspension cell lines. Luciferase activity was measured after 24 hours figures 24. This protocol is for transfecting plasmid dna into mammalian cells. Lipofectamine 3000 reagent maintains a high transfection efficiency within a robust dynamic range of lipid doses for quick and easy optimization. The intracellular trafficking mechanism of lipofectaminebased transfection reagents and its implication for gene delivery. Transient plasmid dna transfection into hela and hacat. Transfection protocol of lipofectamine rnaimax can be obtained from the invitrogen website. Lipofectaminea 3000 transfection reagent delivers better transfection performance than lipofectaminea 2000 reagent as demonstrated by a customer rui eduardo castro, phd, university of lisbon.
In biontex experiments gfp positivity increased to 90. 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. Transfecting plasmid dna into lncap cells using lipofectamine. Transfection protocol lipofectamine 3000 transfection reagent. Add 100l of optimum to the eppendorf tube to dilute the dna and mix by tapping. 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. Jul 17, 2007 lipofectamine 2000 is a lipid based transfection reagent for eukaryotic cells. Cloned genes can be transfected into cells for biochemical characterization, mutational analyses, investigation of the effects of gene expression on cell growth, investigation of gene regulatory elements, and to produce a specific protein. We also provide some tips for optimizing transfection conditions in cultured cells. Lipofectamine 3000 provided gentle but efficient transfection in several line of interest to our lab, heioc1 mouse cochlear 69fold, nccit and ntera2 3 fold testicular cancer lines and allowed for very nice dose response assays for cisplatin sensitvity assays. May 11, 2016 the intracellular trafficking mechanism of lipofectamine based transfection reagents and its implication for gene delivery. Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations. Add 6l of lipofectamine2000 from biostores and 100 l of.
General considerations for using the transfex transfection reagent. Lncapspecific transfection reagent is commercially available from altogen biosystems. Conditions were used according to the manufacturers recommendation for both lipofectamine 2000 and for jetprime. In vivo transfection reagents delivery to a specific animal tissue or organ is. If designing an experiment that involves transfection, ensure that setup coincides with a cell passage. Each reagent was used to transfect hek 293, hela, lncap, a549, and. Enable high transfection efficiency in novel genome editing applications. Lipofectamine 2000 transfection, the expression of gfp was detected in 76,4% of hela cells with a mean fluorescence intensity of 437. Protocol 1 stable transfection using lipofectamine 2000 in 6 well plates. It is best to use polypropylene instead of polystyrene tubes.
Cells in a 6 well dish, plated and at 70% confluence. Optimized conditions for successful transfection of human. 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.
The media needs to be changed at least 1hr prior to transfection. Transfecting plasmid dna into lncap cells using lipofectamine ltx reagent. With lipofectamine 3000, do i need to remove lipofectamine. The day before transfection, dissociate cells that are 8090% confluent in a t75 flask. Lipofectamine 2000 cd reagent complexes can be added directly to cells in culture medium. Genjet in vitro dna tranfection reagent for lncap cells from signagen.
We appreciate greatly the technical service of thermofisher and find them an. Reagent, wv, was used along with the lipofectamine 3000 transfection reagent for all transfections. The weight ratio of lipofectamine 2000 to pdna was kept constant at 3. Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1.
Transfection and genome engineering fisher scientific. Count the cells using standard trypan blue exclusion. Transfection efficiency of p19 cells is between 3050% from 5099% for many other stable cell lines. Transfection protocol lipofectamine 3000 transfection reagent lncap prostate cancer cells complete growth medium component cat. Invitrogen lipofectamine 2000 transfection reagent fisher. This reference provides a recommended procedure to transfect plasmid dna into lncap human prostate epithelial carcinoma cells atcc cat. Lipofectamine 2000 reagent protocol 202lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. In hacat cells, lipofectamine 2000 achieved a transfection efficiency of 22. Origin of the lncap cell line the lncap cell line was established in 1977 by j. Exp, transfection efficiency based on expression of the transfected luciferase gene.
Altogen biosystems offers nanoparticlebased transfection reagent kits for the lncap cell line. Transfection guide overview of transfection methods. I want to try to use hela cell and lipofectamine 2000 to do. Atcc achieved transfection efficiencies of 95%, using the protocol. Genscript recommends using lipofectamine 2000 for all transfections. The mechanism of lipofectamine 2000 mediated transmembrane. Lipofectamine 2000 dna transfection reagent protocol. Transfection reagents are highly efficient for dna and sirna transfection in vivo and in vitro. The lncap prostate cancer cell line is widely used in clinical oncology research. For additional information on scaling your transfection reaction, see page 4.
I wondering how much dna i should use for stable transfection with lipofectamine 2000 tranfection reagent. Crl1740 is an epithelial, prostate carcinoma cell line. To optimize the amount of lipofectamine 2000 for transfection in a 24well plate, start. B hela cells were transfected with luciferase encoding plasmid dna using either transit.
Atcc achieved transfection efficiencies of 95%, using the protocol described below. Du145 transfection kit prostate carcinoma cells altogen. Plate cells for transfection only 1 day before the experiment. Lipofectamine 2000 reagent thermo fisher scientific. Experimental method of delivery of foreign molecules rna, dna, proteins into cells is known as transfection. To perform transfection experiments in other cell culture plates, simply multiply the suggested quantities by the relative surface area of your plate. Invitrogen lipofectamine 3000 transfection reagent fisher.
Lipofectamine 2000 transfection reagent from invitrogen. Researchers use lipofectamine 2000 reagent for sirna and shrnabased gene knockdown experiments, as well as for gene expression studies. Each reagent was tested with a range of transfection reagent. Invitrogens lipofectamine 2000 is one of many cationic lipidbased transfection reagents presently on the market. Hek cells were first transfected with calcium chloride protocol and the. I use 24well plates for transfections in triplicates. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. Transfection efficiencies lower than 50% were not reported in this table.
Transient plasmid dna transfection into hela and hacat cells. 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. Invitrogen lipofectamine 2000 transfection reagent. Optimum amount needed is determined from the protocol for lipofectamine 3000 transfection reagent. Transfection protocol lipofectamine 3000 transfection. Transfecting plasmid dna into lncap cells using lipofectamine 3000 reagent.
Max % of positive cells after transfection of a gfpplasmid 3. What transfection reagent do people usually use for lncap. Lipofectamine 2000 transfection protocol 10cm plates reagents. It is recommended that cells are growing at 9095% confluence prior to transfection for adherent cells. Typically 100 ng and 500 ng of plasmid dna were transferred to each well of the 96well plate and 24well plates, respectively. To transfect cells with sirna, follow the protocol as described for dna but. Several cell densities and lipid volumes were investigated to determine optimal transfection conditions, shown by the shaded boxes. Transfection of small molecules and genetic material into cultured mammalian cells is known as in vitro transfection a lncap transfection kit is commercially available see link. Lipofectamine 3000 reagent thermo fisher scientific fr. However, for cotransfection of sirnachimera and plasmid dna, lipofectamine 2000 should be used.
Via, cell viability assessed by the celltiterfluor cell viability assay compared to a notransfection control. 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. Do any one has protocol for stable transfection with lipofectamine 2000. Enhanced transfection efficiency using lipofectamine 3000.
Invitrogen lipofectamine 3000 transfection reagent 0. Make sufficient 10% fbs in optimem media wo antibiotics sterile filter 2. A transfection enhancer, the 3000 enhancer reagent 1. I have transfected gfpar in to hek293 and lncap cell lines in serum free. The comparative utility of viromer red and lipofectamine for. Lipofectamine contains lipid subunits that can form liposomes in an aqueous environment, which entrap the.
It is considered an appropriate host for studying prostate and hormone functions and is extensively used in drug discovery and different types of biomedical applications. Lncap transfection reagent prostate carcinoma altogen. Lncap specific transfection reagent is commercially available from altogen biosystems. Choosing the right transfection reagent for optimal efficiency. Each reagent was used to transfect hek 293, hela, lncap, hepg2, and a549 cell lines in a 96well format, and gfp expression was analyzed 48 hours posttransfection. A sample protocol is listed here for transfection experiments performed in 6well plates.
The intracellular trafficking mechanism of lipofectamine. Lipofectamine 2000 is a lipid based transfection reagent for eukaryotic cells. Lipofectamine ltx reagent is a proprietary, animalorigin free formulation for the transfection of dna into eukaryotic cells with low cytotoxicity. Transfection guide overview of transfection methods promega. The comparative utility of viromer red and lipofectamine. Lipofectamine 2000 transfection reagent dartmouth college. Lipofectamine 2000 transfection for hek293 cells about 6070% transfection rate transfect cells 6well plate.
Transfex transfection of plasmid dna into lncap fgc cells lncapfgc atcc cat. What percentage confluency should be used for stable transfection. Transfection of nih3t3 cells, hela, swis 3t3, 293t with. Comparative transfection efficiency of jetprime versus its main competitor.
1040 1056 1412 980 320 510 1546 223 1026 1318 1650 915 1440 1071 1260 173 1275 1254 1533 1597 536 289 750 25 367 1537 598 571 558 1182 573 1292 1385 315 1429