Transfection of hek293 cells with lipofectamine 2000 pdf

In 1977, human embryonic kidney cells were transformed with fragments of human adenovirus type 5, giving rise to todays widely used hek293 cell line. Hek293 has since become a popular choice for recombinant protein expression and viral production due to its relatively easy maintenance as well as its amenability to various transfection methods. C wildtype or cgas l929 cells were transfected with htdna or polyi. Can anyone provide me with a proper transfection protocol into hek293 cells with lipofectamine 2000. A guide to transient expression of membrane proteins in hek293. Thermo scientific nunc 24well cell culture treated multidishes 142475 invitrogen lipofectamine 3000 transfection reagent l3000008 gibco optimem i reduced serum medium 31985062 step tube complexation components amount per well 24well. Recombinant proteins expressed in transiently transfected cells can be identified with a method that. Maintain p19 cells undifferentiated in mem with 10% serum. Lipofectamine 3000 that displayed strong toxicity to hek293 cells.

The reagent provides high transfection efficiency in many cells types and formats. I use hek293 cell lines and trying to transfection d3r. Our lipofectamine reagent protocols have been optimized for efficiency, viability, and reproducibility across a broad range of cell types. This protocol isintended for the transfection of adherent mammalian tissue culture cells. Aug 07, 2012 place your 24well plate back into the incubator and grow cells for 1 to 3 days at 37 celsius. B western blot for cgas and gapdh protein levels in wt or cgasl929 cells. An improved method for increasing the efficiency of gene. Transfection efficiency is best if hek293 cells are maintained by splitting cells every 3 days note 2 and 3. Hek293 cells are useful for many transfection experiments, particularly the propagation of adenoviralbased and retroviralbased vectors. Highest transfection efficiency in cells at 90% confluency.

Arpe19 transfection kit retinal pigment epithelium. If there appears to be largescale loss of cells after lipofectamine treatment, then it might pay to try using polylysinecoated plates. Lipofectamine gives 70% positive cells, but seems to be toxic. Carefully transfer the transfection mix to the lentix 293t packaging cells. Protocol 2 producing lentivirus in hek293t cells using a. Lipofectamine transfection of c2c12 cells bridges lab. Hek293 cells, bj fibroblasts, and ecs were transfected with 1. Add the transfection mix dropwise being careful not to dislodge the cells. The bioluminescence system luciferase reporter assay system is widely used to study gene expression, signal. I always use lipofectamine 2000 for transfection of hek293t cells. 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. They are also known, more informally, as hek cells. We did not find substantial transfection efficiency variations in different batches of lipofectamine 2000. The main advantage of using lipofectamine 2000 however, was its ability to transfect a range of very different cell types coupled with a high efficiency of transfection.

Altogen biosystems provides in vivo transfection reagents, over 100 preoptimized in vitro transfection kits for cell lines and primary cells, and electroporation delivery products. Transfections were performed on a549 and hek293 cells following. The transfection medium was then removed and the cells were incubated for an additional 48 h in complete medium 2 ml per well. Perform the transfection in the late afternoon because the transfection mix should only be incubated with the cells for 1215 hours. In my experience lipofectamine works like a charm in many cells, including hek293. Altogen cro offers in vivo rnai services, tumor xenograft models, toxicology testing, stable cell line generation, and. Liposome lipidbased formulation has the least influence on. Transfection guide overview of transfection methods. Can anyone provide me with a proper transfection protocol. Lipofectamine 2000 reagent thermo fisher scientific.

What are suitable transfection reagents for hek 293 cells. Gently mix media on cells via figure 8 pattern and back and forth motions. Original article an improved method for increasing the. Feb 08, 2010 with the exception of hek293 cells, when compared to chemical dna delivery vehicles, nucleofection appears to be, in general, more effective and less toxic. Cells should be plated on collagencoated dishes, as for normal propagation.

With the exception of hek 293 cell transfection, nucleofection proved to be less toxic and more efficient at effectively delivering dna into the cells as determined by cell proliferation and gfp expression, respectively. In polypropylene microfuge tubes do not use polystyrene tubes, make a cocktail for each transfection. After 1 month of transfection, ags cell lines stably transfected with vegfr12 and nrp1 were screened with 5. Pdf optimization of sw480 colon cancer cells transfection. Plate cells for 212hrs but not over 24 hours prior to transfection to achieve a 4080% confluence at the time of transfection. I agree with parvoman that one of the main considerations is how well the cells are growing. Hek293 transfection reagent is tested functionally by transfection of hek293 cells with a small.

Cells were also assayed for cell viability and compared with a no transfection control for each cell line. Lipofectamine 2000 dna transfection reagent protocol see page 2 to view a typical dna transfection procedure. Transfection efficiencies lower than 50% were not reported in this table. Good news, my antibody works pretty well tested 293t untransfected, transfected 293 two different clones, and psirpg transfected cells transfection control. For example, the therapeutic applications of rnai can be achieved by both transfection agents16 and aav vectors. The following morning, carefully aspirate the media. The current study aims to characterize and determine the nonviral vector based gene transfection efficiency with human induced pluripotent stem cells hipscs derived cardiomyocytes hipsccms. Advanced transfection with lipofectamine 2000 reagent. The day before plate cells at 45x106p100 to get the following day 8090% confluency. Two component lipidbased proprietary formulation optimized for hek293 cells transfection. The gfp fluorescence intensity in hek293 cells transfected by the lplndna complexes np10 was about 37. Transfection is the process of introducing nucleic acids into eukaryotic cells by nonviral methods.

Maintain p19 cells undifferentiated in mem with 10% serum 7. After incubating your cells, assess the transfection efficiency in each well by viewing gfp fluorescence. Protocol 2 producing lentivirus in hek293t cells using a 2nd generation lentiviral system. Plate cells at least 12hrs but not over 24 hours prior to transfection to achieve a 4080% confluence at the time of transfection. Transfection efficiencies were optimal around 40% of transfected cells per spot with 11.

Hek293 is a cell line derived from human embryonic kidney cells grown in tissue culture. Wash the cells 2x pbs seed 10 6 cells in 300 ul warm optimem in 24 wellsplates. Request pdf advanced transfection with lipofectamine 2000 reagent. Hek293 cells were transiently transfected with lentivirus expression vector plenti cmvgfpdest separately using lipofectamine 3000, 2000 or. Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. 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. Use a range of several ul of lipofectamine and ug dna to increase viability after transfection and efficiency. Full text highly efficient transfection of human induced. The transfection efficiency and toxicity is equivalent following nucleofection or lipofectamine transfection of hek293 cells figure figure1 1. 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. Lipofectamine 2000 dna transfection reagent protocol. Lipofectamine 2000 from invitrogen biocompare product. Plate cells so they will be 7090% confluent at the time of transfection.

Prewarm 50ml of optimum stored in cold room at 4c 10min. 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. Transfection commonly refers to the introduction of nucleic acids into eukaryotic cells, or more specifically, into animal cells. All amounts and volumes are given on a per well basis. The human embryonic kidney 293 hek293 cells are commonly used as host for the heterologous. 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 day before transfection, plate cells in 500 l of growth medium without antibiotics such that they will be 3050% confluent at the time of transfection. Following transfection the cells were incubated at 37c in humidifiedair 5% co 2 for 2 h. My main purpose is to show that my antibody for bob works. Transfection of hek293 cells using three different reagents trojanporter lipofectamine 2000 nanofectamine summary protocol the cells were plated the day prior to transfection. For additional information on scaling your transfection reaction, see page 4. So i did the tranfection, followed up by western blotting. I want to do a transfection to overexpress protein bob in 293t cells. Unfortunately, reagenst like fugene6 and translt1 give only 510% efficiency.

Transient transfection of mammalian cells with lipofectamine. L reverse transfection of rnai reverse transfection is faster to perform than forward transfection and is the method of choice for highthroughput transfection. Transfection protocol transfection components component cat. Three alleles of cgas gene in l929 each has a frameshift deletion. Invitrogen lipofectamine ltx reagent is a proprietary, animalorigin free formulation for the transfection of dna into eukaryotic cells with low cytotoxicity. For instance, in a recent study, bernard and colleagues achieved successful mir217 mimic transfected to hek293 cells via lipofectamine 3000 only treatment. Prior to transfection, hek 293 cells are preincubated with serumfree dmem medium for 1 h. If you find this doesnt work for your specific cell type, then you can you look to our cell specific protocols for further optimization. Transfection guide overview of transfection methods promega. Higher densities can lead to spontaneous differentiation. Lipofectamine 2000 cd reagent complexes can be added directly to cells in culture medium. Systematic screening of commonly used commercial transfection. 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 and suspension cell lines.

Hek 293 cells are efficiently transfected with lipofectamine, with typically 70% of cells showing fluorescence following transfection with green fluorescent protein gfp. Using various chemical or physical methods, this gene transfer technology enables the study of gene function and protein expression in a cellular environment. I need high efficiency of transfection in order to do western blot on transfected cell lysates. The regulatory mechanism and biological significance of. 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. I have used abms dnafectin on hek293 cells and had no issues with it. Lipofectamine 2000 invitrogen was used to transfect the plasmid into gc cells. 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.

Thee two most important factors in choosing transfecting reagent are. Transfection reagents are highly efficient for dna and sirna transfection in vivo and in vitro. Perform reverse transfection by preparing complexes inside the wells, and then adding cells and medium. Lipofectamine ltx dna transfection reagent protocol see page 2 to view a typical plasmid transfection procedure. Transfection of nih3t3 cells, hela, swis 3t3, 293t with. We compared the transfection efficiency of hipscs with that of human embryonic kidney hek 293 cells. Laboratory 2010 lipofectamine 2000 transfection protocol 10cm plates reagents.

Incubate the cells for 18 h, or until the following morning. Hi, i tried to transfect my hek293t cells with pegfp using lipofectamine 3000, following manufacturers instructions. Transfection is the introduction of dna, rna, or proteins into eukaryotic cells and is used in research to study and modulate gene expression. Each transfection was carried out in triplicate and repeated 2 to 3 times. Hek293 transfection reagent epithelial kidney cells. These cells were developed from embryonic kidney cells, and are useful in a variety of molecular and biological research, many of which have medical applications. Thus, transfection techniques and protocols serve as an analytical tool that facilitates the characterization of genetic functions, protein synthesis, cell. However, i could only reach at most 2030% transfection efficiency. Nonviral vector based gene transfection with human induced.

Add 100l of optimum to the eppendorf tube to dilute the dna and mix by tapping. Hek293 transfection reagent is supplied in liquid form at a concentration of 0. Transfection p19 cells with lipofectamine 2000 the same protocol can be used for 293t cells 1. The transfection efficacy of lipofectamine 2000 was compromised by. Polyplustransfection 2019 brochure chemie brunschwig. Optimizing transfection lipofectamine 2000 cd reagent. In addition to calcium phosphate, i have also used lipofectamine and genejuice all have demonstrated very high transfection. This is often the best place to start, especially in a new cell line. Further analysis of the transfected hipscs showed that the differentiation of hipscs to hipsccms was not altered by nps. Highest transfection efficiency in many cell types and formats. This is necessary because serum is replete with rnases that degrade. General procedure for transfection of mammalian cells with. Original article an improved method for increasing the efficiency of. I have ordered lipofectamine 2000 from thermo fishers scientific.

The animal originfree formulation ensures that mammalian cell culture and bioproduction processes are free of animalderived materials. Highest transfection efficiency in many cell types and formats e. Transfection protocols thermo fisher scientific in. Lipofectamine 2000 transfection for hek293 cells about 6070% transfection rate transfect cells 6well plate. Scaling up or down lipofectamine 3000 reagent transfections. Lipofectamine 2000 transfection reagent from invitrogen.

I use 24well plates for transfections in triplicates. The cell culture must have 90% viability and be 85% confluent on the day of transfection. Similarly, in this study, we achieved an acceptable transfection efficacy 18. Cells in a 6 well dish, plated and at 70% confluence. Add 6l of lipofectamine2000 from biostores and 100 l of. If you let them reach confluency, even if this happened a few passages ago, the transfection efficiency will be significantly diminished. Hek293 cells were transiently transfected with lentivirus expression vector plenticmvgfpdest separately using lipofectamine 3000, 2000 or. If cells are not at the right confluence, do not wait until the next day to perform transfection, as this can significantly affect transfection efficiency. Lipofectamine and nucleofection of hek 293 were essentially equivalent in terms of toxicity and efficiency. This reference provides a recommended procedure to transfect plasmid dna into hek 293, human embryonic kidney cells atcc no. Theres a difference between the two protocols lipofectamine 3000 has an extra reagent that needs to be added. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. Even though cell lines differ in their experimental properties, this protocol will yield efficient transfection at low nano molar concentrations in most of the commonly. To transfect cells with sirna, follow the protocol as described for dna but.

Here we report a method for efficient transfection of in vitrotranscribed mrna into two different types of human adherent cells, the neuroblastoma cell line sknas, and the transformed kidney cell line hek293. In addition to being reliable hosts for transfection, hek293 cells are particularly helpful for the production of therapeutic proteins as well as viruses for gene therapy. By using newly trypsinized adherent cells in suspension and lipofectamine 2000, we detected a transfection efficiency of 8090% in both. The glosensor camp hek293 cell line was maintained for up to 20 passages on 10cm culture dishes in dmem growth medium containing 50. Lower confluence will achieve higher percentage of transfection but may yield a lower absolute number of transfected cells. Optimized conditions for successful transfection of human. Transfection protocol lipofectamine 3000 transfection. Using cx43 shrna lentivirus combined with lipofectamine 3000 transfection reagent, we can achieve about 90% cx43 knockdown efficacy in huvecs. Trojanporter was used as described in the protocol for a 6well plate. A sample protocol is listed here for transfection experiments performed in 6well plates. At 48 h after transfection of the plasmid containing hpv16 genome into hek293t cell line, hpv16 vlps were observed from the cells with an electron micrograph, the virus dnas existed in hek293 cells for at least 60 h, and the fresh hpv16 dnas were synthesized at 20 h after transfection. Lipofectamine 2000 cd reagent is a proprietary animal originfree formulation for transfecting nucleic acids into eukaryotic cells. To perform transfection experiments in other cell culture plates, simply multiply the suggested quantities by the relative surface area of your plate.

For some cell types, such as cos7, the transfection efficiency was over 95% while other hardertotransfect cells, such as caco2, displayed efficiencies around 75%. Cells and molecular biology research methods, protocols, and lab techniques. Increased transfection efficiency by the directed transport, especially for low amounts of nucleic acids high transfection rates for adherent mammalian cell lines and primary cell cultures suspension cells and cells from other organisms also successfully transfected but need to be immobilized mild treatment of cells. In forward transfections, cells are plated in the wells, and the transfection mix is generally prepared and added the next day.