What i found is lipofectamine 2000 is toxic to cells, so i reduced the recommended quantity of the invitrogen protocol. Pei complex is endocytosed by the cells and the dna released into the cytoplasm sonawane et al. We found that the most successful approach was to add the dna transfection mix to 80% confluent hela cells for 5 hours then gently remove. 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. Hela transfection kit cervix adenocarcinoma altogen. The culture medium was replaced with 2 ml of fresh serumcontaining medium before transfection. Lipofectamine ltx reagent is a proprietary, animalorigin free formulation for the transfection of dna into eukaryotic cells with low cytotoxicity. Our lab has been satisfied with the routine use of the transithelamonster transfection kit. Transfection and selection data of hela cells by various antibiotics in differing media. Hela cells morphology 24 hours after transfection by fugenehd, fugene6 and competitor lipofectamine 2000 click here to view the protocol control fugene6 untransfected cells reagent dna ratio 0. Peitransferrinfection kit thermo fisher scientific. Transfections exhibit high target protein expression with very little cell toxicity. May 18, 2000 a variety of methods have been developed for the transfection of cells, including electroporation 1,2, lipofection 3,4, calcium phosphate coprecipitation 5,6 and deae dextran 7,8.
Apr 08, 2010 how many 293t or hela cells should i seed for transfection in 10cm dishes i want to harvest after 48h. Transfection conditions should be optimized for each cell type to ensure maximal gene expression with minimal cellular toxicity. I know that if you have a selective medium with antibiotics puromycin, g418 etc. Transfection starts 23 days after seeding the cells 70%. Transfection of cells at lower densities often leads to cellular toxicity because of an increase in the number of complexes delivered per cell. Dna can be introduced into a host cell by transfection with polyethylenimine pei, a stable cationic polymer boussif et al. Evaluation of the effects of amphiphilic oligomers in pei based. General protocol for transfection of stem cells, primary cells, and continuous cell lines with atcc transfex transfection reagent. Method to enhance transfection efficiency of cell lines and. Many variations and applications of transfection exist. General protocol for transfection of stem cells, primary. Hela s3 cells were seeded into 35mm culture dishes 4.
I performed polyethyleniminebased transfection in hek293 cell line with an inhouse protocol which worked. We report a method that allows for a 29fold increase in transfection efficiency compared to the standard cationic lipidbased protocol. The method involves a brief incubation of freshly trypsinized cells with transfection complexes, followed by incubation in cell growth medium containing serum and antibiotics. Pulsed electrical fields can be used to introduce dna into a wide variety of animal cells 1,2. Understanding the impact of transfection mediated toxicity is important for every researcher. Mirus broad spectrum transfection reagents, transitlt1 and transit2020 outperform lipofectamine 2000. But unfortunately the survival rate of cells has only been 46,9 61,9 %. Pdf a simple and rapid nonviral approach to efficiently transfect. I have used both fugene and jetprime as transfection reagents in hela cells. There are many factors that can influence transfection efficiency, a number of which are specific to the target cell. Sep 17, 2012 the optimal confluency at the time of transfection is 60% to 80% for adherent cells. You can not use antibiotics during transfection step because it will cause cell death. Since their initiation, hela cells have been used in transfection experiments all over the world to study viruses. Transfection of dna into eukaryotic cells is a common method to study biological mechanisms.
A major goal is the efficient and specific delivery of genes into the desired target cells. The gene transfection efficiency and cell cytotoxicity of pei are proved to be closely. Also, hela cells were used by jonas salk to test the first polio vaccine in the 1950s. Two types of transfection, transient and stable, are used experimentally.
Vero cells became vacuoled if media was not changed 24h post transfection, the remaining cell types were fine with or without a media change. Transfection of mammalian cells with fluorescent protein fusions. The internalization route resulting in successful gene expression. Is polyethylenimine pei transfection reagent suitable in the hela or a549 cell lines. Some researchers have shown that cellular uptake of pei polyplexes affects other cellular. For electroporation of hela cells, this protocol has been successful with a mixture of 2. Costeffective gene transfection by dna compaction at ph 4. Transfection of mammalian cells using linear polyethylenimine. Impact on biological research and informed consent duration. Primary cell transfection while immortalized cell lines traditionally have served as the common targets of transfection, primary cells are often preferred as they more closely resemble the invivo nature of tissues. Add 2g of dna to a sterile eppendorf, along with 250l of buffer ec. Prepare dna according to the manufacturers instructions for the transfection procedure. We have developed a simple protocol to transfect mammalian cells using linear polyethylenimine pei.
Htb22 is an epithelial, breast adenocarcinoma cell line that was derived from a metastatic pleural effusion. To test the cell cycle dependence of transfection with linear pei, we elutriated and transfected logarithmically growing hela cells. This reference provides a recommended procedure to transfect plasmid dna into hek 293, human embryonic kidney cells atcc no. For hela cells we have already achieved a transfection efficiency of 87, 8 97,8 % with lipofectamine. Hela cells were transfected with a gfp encoding plasmid dna using either transitlt1, transit2020 or lipofectamine 2000 at reagenttodna ratios of 3. Buy hela cells of henrietta lacks by singh, nishi isbn. Jul 21, 2015 pei transfections with gfp were equivalent or better than fugene in all 4 cells types, in particular hela cells. Hela cells morphology 24 hours after transfection by fugene. Cells remain viable posttransfection and can be readily infected with virus without any problems.
Transfex transfection of plasmid dna into mcf7 cells. Important guidelines for transfection follow these important guidelines when transfecting dna into hela s3 cells using lipofectamine ltx reagent. Transfection is the process of introducing nucleic acids into eukaryotic cells by. Protocol for hela cell culture and plasmid transfection av02hela cell culture 1. General considerations for using the transfex transfection reagent. Principles and mechanisms of mammalian cell transfection. It was the hela cell line that took up the polio virus and.
Transfection is employed in a variety of research fields. Transfection is the introduction of dna, rna, or proteins into eukaryotic cells and is used in research to study and modulate gene expression. Transfection of mammalian cells by electroporation nature. Hela is also receptive to human adenovirus 3, encephalomyocarditis virus and human polioviruses 1, 2, and 3. Gene delivery by hydroxyapatite and calcium phosphate. Atcc achieved transfection efficiencies of 8590%, using the protocol described below. Protocols for transfecting the most common cell lines with x. Hela tests positive for keratin and lysophosphatidylcholine. I do have cell toxicity, but not so serious as yours. Effectene transfection of hela cells qiagen transfection solution 1. Although a wide panel of techniques and vectors viral and nonviral have been developed that work with variable efficiency, most vectors lack a target cell specificity. Hela transfection information and resources hela cell line. Our laboratory uses pei over other cell transfection reagents because of its low cost.
Everyday low prices and free delivery on eligible orders. Add 16l of enchancer solution, vortex and let stand for 5 minutes at room temperature. Lipofectamine is great, but its a generic that can have trouble sometimes with picky cells. Transfection, technique used to insert foreign nucleic acid dna or rna into a cell, typically with the intention of altering cellular properties. Pei condenses dna into positively charged particles that bind to anionic cell surfaces. A, b, g, h huh7, c, d cos7, and e, f, i, j hela cells were pretreated. In general, i used fly extracts to help the cells but meanwhile found out that i can do without and still get about 50% transfection measured as gfp positive cells in facs. It may also refer to other methods and cell types, although other terms are often preferred. Electroporation protocol for hela cells transfection protocol protocol no. Thus, transfection techniques and protocols serve as an analytical tool that facilitates the characterization of genetic functions, protein synthesis, cell growth and development. Protocol for hela cell culture and plasmid transfection av.
Livecell imaging to compare the transfection and gene. Co transfection can allow a researcher to study the effect of missense mutations on the function of cellular proteins. Spontaneous transfection of mammalian cells with plasmid dna vania l. Even though hela cells are very hardy, the hela cell line is fairly easy to transfect and very receptive to viral vectors, making it a popular research tool in virology. Transfection guide overview of transfection methods promega. Electroporation works well with cell lines that are refractive to other techniques, such as calcium. Live cell imaging microscopy of hela cells during transfection with capdnacap pei nanoparticles and lipofectamine. Hela cells are also known to contain human papillomavirus 18 sequences. Transfection is the process of deliberately introducing naked or purified nucleic acids into eukaryotic cells. Spontaneous transfection of mammalian cells with plasmid dna.
Add 25l of effectene solution, vortex and let stand for 5 minutes at room temperature. There are now many strains of hela cells, but all are descended from the same tumor cells removed from lacks. Transithelamonster transfection kit research reagent products. High passage of hela cells and use of antibiotics or growth factors may require using larger volumes of hela transfection reagent per reaction.
Transfection efficiency of pei polyplexes in the presence of endocytosis inhibitors. Maximova institute of molecular biology, bulgarian academy of sciences, 11 sofia, bulgaria. Cellular uptake mechanism of nonviral gene delivery and means. We found that the most successful approach was to add the dna transfection mix to 80% confluent hela cells for 5 hours then gently remove the media and replace with the normal serum containing. Hi, our viromer red is a peiderived reagent and works perfectly fine with hela and a549cells 6570% of efficiency for plasmid transfection, 80% for mrna. Despite the ability of pei to transfect a wide range of cell types. To obtain the highest transfection efficiency, optimize transfection conditions by varying hela cell density and amount of transfection reagent.
Antibody quantities 7 days post transfection expressed by hek293 and proprietary hekinv. A preoptimized hela transfection kit is available from altogen biosystems, which includes. Xgal staining of raw cells 24 hours after transfection. I am doing transfection using lipofectamine 2000 and plasmid dna, too. Is polyethylenimine pei transfection reagent suitable in. Hekinv is an optimized host cell line for tge processes generated by a directed evolution approach resulting in a 3 fold increase in productivity in comparison to the parental host cell line. Greater than 90% of xdc293 cells and 98% of hela cells transfected using our method were positive for egfp expression as determined by flow cytometery. Hela cells were seeded the day before transfection with the density 1. Invitro cytotoxicity of polyethyleneimine on hela and vero cells. Once cells have been transfected, various methods can be used for analysis post transfection and for assessing transfection efficiency. Here, rnai was transfected into hela cells in order to downregulate the endogenous brca1 protein, which causes a reduction in the number of gfp positive cells. Furthermore, schneider cells are slow, thus harvesting cells after 72 h instead of after 48 h post transfection can be beneficial.
Low toxicity in hela cells transfection mirus bio llc. We found that the most successful approach was to add the dna transfection mix to 80% confluent hela cells for 5 hours then gently remove the media. Transfection efficiency was tested on the hela cells which were treated with. Sep 05, 2012 mammalian cell transfection is a technique commonly used to express exogenous dna or rna in a host cell line for example, for generating rnai probes. There are many different ways to transfect mammalian cells, depending on the cell line characteristics, desired effect, and downstream applications.
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