Transfection of hek293 cells with lipofectamine 2000 manual

My main purpose is to show that my antibody for bob works. Can anyone provide me with a proper transfection protocol. Two component lipidbased proprietary formulation optimized for. Lipofectamine 2000 transfection for hek293 cells about 6070% transfection rate transfect cells 6well plate. I previously believed that the best method was using lipofectamine 2000 with plus reagent and an overnight incubation with optimem.

Hek293 transfection reagent is tested functionally by transfection of hek293 cells with a small. Strong expression of egfp was observed in hek293 cells, mouse. Divide the cells 1824 hours before transfection to ensure that the cells are actively dividing and reach the appropriate cell density at the time of transfection. Transfection protocol lipofectamine 3000 transfection reagent lncap prostate cancer cells complete growth medium component cat. Using hek293t expression system to study photoactive. Plate cells so they will be 7090% confluent at the time of transfection. The lipofectamine 3000 is the bees knees though, i managed to get loads of transfects from. Increase transfection efficiency and cell viability with.

The expifectamine 293 transfection kit is a core component of the expi293 expression system. I have been doing research with primary rat cardiomyocytes for more than seven years, and plasmid transfection of these cells is not easy. Two component lipidbased proprietary formulation optimized for hek293 cells transfection. In both cases, cells are still actively growing when harvested. After incubating your cells, assess the transfection efficiency in each well by viewing gfp fluorescence. Optimized conditions for successful transfection of human. Mir2304 and lipofectamine 2000 transfection reagent life technologies, cat. Plate cells at least 12hrs but not over 24 hours prior to transfection to achieve. This protocol is for transfecting plasmid dna into mammalian cells. What are suitable transfection reagents for hek 293 cells. 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.

Add 6l of lipofectamine2000 from biostores and 100 l of. Aug 07, 2012 place your 24well plate back into the incubator and grow cells for 1 to 3 days at 37 celsius. Mar 03, 2014 the transfection of cells already with 0. Then, cells were washed with pbs twice and transduced with scaav2cbapegfp at 2,000. For proteins, such as antigens, hek2936e, and hkb11 cells gave in most instances the best results, while for expression of antibodies, transfection into hek293 t cells frequently resulted in higher. Plate cells for 212hrs but not over 24 hours prior to transfection to achieve a 4080% confluence at the time of transfection. Cut plasmid with an appropriate restriction enzyme to produce a linear fragment for transfection.

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. Transfection protocol lipofectamine 3000 transfection. An improved method for increasing the efficiency of gene. Higher densities can lead to spontaneous differentiation. I have ordered lipofectamine 2000 from thermo fishers scientific. Efficiently transfect difficulttotransfect cells lipofectamine 3000 reagent is designed to efficiently transfect difficulttotransfect cells, yielding superior transfection performance across the broadest array of. Lipofectamine ltx reagent is a proprietary, animalorigin free formulation for the transfection of dna into eukaryotic cells with low cytotoxicity.

In a reverse transfection protocol, cells are added directly to a plate. Hi, i tried to transfect my hek293t cells with pegfp using lipofectamine 3000, following manufacturers instructions. According to the manufacture manual, pcr products were first mixed with 10. Transfection protocol lipofectamine 3000 transfection reagent lncap prostate cancer cells. The human embryonic kidney 293 hek293 cells are commonly used as host for. To perform transfection experiments in other cell culture plates, simply multiply the suggested quantities by the relative surface area of your plate. Highest transfection efficiency in many cell types and formats e.

Nov 20, 2008 in my experience lipofectamine works like a charm in many cells, including hek293. For additional information on scaling your transfection reaction, see page 4. Theres a difference between the two protocols lipofectamine 3000 has an extra reagent that needs to be added. Similarly, in this study, we achieved an acceptable transfection efficacy 18. Our linear pei protocol is as effective as commercial reagents in the transfection of hela cells and xdc293 cells, a derivative of hek293 cells, but at a fraction of the cost. I want to do a transfection to overexpress protein bob in 293t cells. Lipofectamine 2000 dna transfection reagent protocol see page 2 to view a typical dna transfection procedure. To prepare hek 293t cells for reverse transfection. Transfection guide overview of transfection methods. The transfection reagent, lipofectamine2000, was selected for. 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.

Our linear pei protocol is as effective as commercial reagents in the transfection of hela cells and. Wash the cells 2x pbs seed 10 6 cells in 300 ul warm optimem in 24 wellsplates. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. Maintain p19 cells undifferentiated in mem with 10% serum 7. A sample protocol is listed here for transfection experiments performed in 6well plates. Transfection can be performed both in the presence or absence of serum. Add dnalipofectamine complexes 500 ul to the 2 ml of media on the cells. Original article an improved method for increasing the efficiency of. Protocol 1 stable transfection using lipofectamine 2000 in 6 well plates. Transfection protocol lipofectamine 3000 transfection reagent. Expression in the presence of various transfection reagents a hela cells were treated with different transfection reagents for 6 hr without plasmids, according to the companys manual. Hek293 transfection reagent epithelial kidney cells. Thus, hek293 is an excellent cell line to use in transfection experiments or to produce.

Transfection p19 cells with lipofectamine 2000 the same protocol can be used for 293t cells 1. Maintain the same seeding conditions between experiments. Lipofectamine 2000 is that it has an extensive, very useful manual. 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. Maintain p19 cells undifferentiated in mem with 10% serum. Lipofectamine transfection of c2c12 cells bridges lab. Lipofectamine ltx dna transfection reagent protocol see page 2 to view a typical plasmid transfection procedure. Prewarm 50ml of optimum stored in cold room at 4c 10min. Transfection is the process of introducing nucleic acids into eukaryotic cells by nonviral methods. Place your 24well plate back into the incubator and grow cells for 1 to 3 days at 37 celsius. If you let them reach confluency, even if this happened a few passages ago, the transfection efficiency will be significantly diminished. In the present study, we screened a large set of transfection reagents using relatively easy and hardtotransfectcelllines and identi. 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.

The transfection medium was then removed and the cells were incubated for an additional 48 h in complete medium 2 ml per well. Systematic screening of commonly used commercial transfection. We have developed a simple protocol to transfect mammalian cells using linear polyethylenimine pei. Transfecting plasmid dna into hek 293 cells using lipofectamine. Ive plated cells in appropriate numbers, followed the lipofectamine 2000 protocol. Cells should be plated on collagencoated dishes, as for normal propagation. It is designed for transient transfection of high density cultures of hek human embryonic kidney 293 cells. This reference provides a recommended procedure to transfect plasmid dna into hek 293, human embryonic kidney cells atcc no. Transfection efficiencies lower than 50% were not reported in this table. On the day of transfection just prior to preparing complexes. 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. Protocol 1 stable transfection using lipofectamine.

Invitrogen lipofectamine ltx reagent is a proprietary, animalorigin free formulation for the transfection of dna into eukaryotic cells with low cytotoxicity. I agree with parvoman that one of the main considerations is how well the cells are growing. Add 100l of optimum to the eppendorf tube to dilute the dna and mix by tapping. A guide to transient expression of membrane proteins in hek293. Use highly purified, sterile, and contaminantfree dna for transfection. Shop online for a wide selection of invitrogen lipofectamine 3000 transfection reagent lipofectamine 3000 transfection reagent leverages our most advanced lipid fisher scientific. I spent about 5 months trying to optimize transfection protocols of 4 different cell lines including some very hard to transfect cells. General procedure for transfection of mammalian cells with. The hek293 cells cotransfected with cry2cib1 or cry2spa1 were. For instance, in a recent study, bernard and colleagues achieved successful mir217 mimic transfected to hek293 cells via lipofectamine 3000 only treatment. Please refer to the product datasheet for complete protocol details.

Rnai in cultured mammalian cells using synthetic sirnas ncbi. I previously believed that the best method was using. Transfection and expression experiments may be performed directly in expi293 expression medium without the need for media change. Transfection of mammalian cells using linear polyethylenimine. Using hek293 cells, fugene hd reagent produced the most.

Lipofectamine 2000 reagent for highly efficient delivery of dsrna oligomers to a wide variety of mammalian cells for more information about each reagent, see pages 23. Lipofectamine 2000 transfection reagent from invitrogen. I use 24well plates for transfections in triplicates. Follow these important guidelines when transfecting hek 293 cells using lipofectamine ltx reagent. The protocol below describes the transfection of hek293 cells in serumfree hek transfection media. Using various chemical or physical methods, this gene transfer technology enables the study of gene. Uses for the blockit transfection kit use lipofectamine 2000 for highly efficient delivery of. Lipofectamine 2000 was also reported in u2os cells. Thee two most important factors in choosing transfecting reagent are. Each transfection was carried out in triplicate and repeated 2 to 3 times. Can anyone provide me with a proper transfection protocol into. For proteins, such as antigens, hek293 6e, and hkb11 cells gave in most instances the best results, while for expression of antibodies, transfection into hek293 t cells frequently resulted in higher expression levels geisse, 2009.

In summary, it is certainly worthwhile to try parallel expression in several hek293 derived cell lines in order. Liposome lipidbased formulation has the least influence. If there appears to be largescale loss of cells after lipofectamine treatment, then it might pay to try using polylysinecoated plates. 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. I found many cells detach easily after 6h incubation with lipofectamine. Combine the equal volumes of optimemdna and optimem lipofectamine. In a reverse transfection protocol, cells are added directly to a plate containing the.

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. However, i could only reach at most 2030% transfection efficiency. Versatileone reagent for dna, rna, and cotransfection. Good news, my antibody works pretty well tested 293t untransfected, transfected 293 two different clones, and psirpg transfected cells transfection control. After incubating your cells, assess the transfection. After 4h aspirate media and refeed cells with normal media. Alternatively, 5x106 cells can be cultured for 2 days before transfection.

Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. Improved cell viabilitygentle on your cells, with low toxicity. If there appears to be largescale loss of cells after lipofectamine. I have used calcium phosphate method and lipofectamine 3000 to transfection hek 293t cells. Following transfection the cells were incubated at 37c in humidifiedair 5% co 2 for 2 h. For more information about stealth rna, see page 3. Hek293 cells detach after transfection scientist solutions. Starrlab transfection using lipoectamine transfection using lipoectamine. Lipofectamine 2000 is used to deliver the sirnas into hek293 cells.

For example, one reagent may work well with hek293 cells, but another. Optional the final cell density for transfection protocol varies depending on the purpose of experiment. Add dna lipofectamine complexes 500 ul to the 2 ml of media on the cells. In my experience lipofectamine works like a charm in many cells, including hek293. Transfection and expression experiments may be performed. Transient transfection of mammalian cells with lipofectamine. Improved delivery of cas9 proteingrna complexes using. 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. Hek293 cells are easy to grow in culture and transfection kits are commercially available. Plate cells for 212hrs but not over 24 hours prior to transfection to. Lipofectamine 2000 dna transfection reagent protocol.

To perform transfection experiments in other cell culture plates, simply multiply the. Lipofectamine reagent thermo fisher and its newer derivatives are widely used for. Invitrogen lipofectamine 3000 transfection reagent. Plate cells for transfection only 1 day before the experiment. Combine the equal volumes of optimemdna and optimemlipofectamine. 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. For me, fugene and lipofectamine 3000 not 2000 worked the best, while the worst for me were pei and metafectene. So i did the tranfection, followed up by western blotting.