If the medium becomes very dense with cell debris, change to fresh antibiotic medium after 47 d. The table below contains a listing of the cell lines and primary cells successfully electroporated with the neon transfection system or the predecessor instrument the microporator mp100. Neon electroporation transfection thermo fisher scientific us. Establishing transfection methods that enable highly efficient dna uptake has become increasingly important. Monitor the needle to make sure there are no sparks arcing, which will ruin the transfection. Over 4000 publications, cell lines and primary cells available. Universal transfection reagent is used for transient and stable transfection of nucleic acids into various eukaryotic cell lines and hardtotransfect primary cells. Neon transfection system 10 l kit thermo fisher scientific. Transfection reagents are highly efficient for dna and sirna transfection in vivo and in vitro. Neon transfection system instructions for using the neon transfection system for transfection of mammalian cells are described below. Oncogenic gene fusion fgfr3tacc3 is regulated by tyrosine.
Altogen biosystems provides in vivo transfection reagents, over 100 preoptimized in vitro transfection kits for cell lines and primary cells, and electroporation delivery products. Broad spectrum dna delivery achieve high expression in many cell types, including hard to transfect cell lines. Neon transfection system enables fast and efficient delivery of nucleic acids into all mammalian cell types, including primary, stem, and difficulttotransfect cells. Neon transfection of primary mouse t cells the ansel lab. Reverse transfection is defined as the process of adding the encapsulated cargo molecule plasmid dna, sirna, mirna while simultaneously plating the cells. Transfection protocols vary based on cell type to be transfected, transfection method, and transfection reagent. Transfection of h9 is efficient and the resulting cells have a high viability. This device have an electrode in the tip, increasing the rate of viability of cells and the % of transfection.
These methods have advanced to make it possible to deliver nucleic acids to specific subcellular regions of cells by use of a precisely controlled lasermicrocope system. This device allows you to transfection directly in the pipette tip, limiting the steps required. Electroporation using neon electroporation from invitrogen. Typically, 24 to 48 hours between transfection and.
Cell transfection database polyplustransfection expertise. The neon transfection system offers open and transparent protocols that are optimized for ease of use. Mar 03, 2015 invitrogen neon transfection system mpk5000 electroporation lot 551. Protocol overview i electroporated 5 million cells with 4 ug of a gfp plasmid using the neon transfection system 100 l kit. Invitrogen neon transfection system vxmpk5000s duration. Madison, wi, march 8, 2011 in addition to reliable high efficiency transfection reagents, mirus bio has expanded its transfection expertise with the development of the reagent agent a new online tool designed to determine the best solution for delivery of any nucleic acid into any cell type, including hardtotransfect. I will start by giving you an overview of the topics that i will cover during todays webinar. The transfection methods are broadly classified into three groups. The neon 10 l transfection system draws 10 l of cells and transfection.
Cell applications, inc 5820 oberlin drive, suite 101 san diego, ca 92121 open mf, 8am5pm pst. Cells that have been transfected using included 10. Use the table below to troubleshoot transfection experimental failure. Using various chemical or physical methods, this gene transfer technology enables the study of gene. Neon transfection system thermo fisher scientific pw. With an extremely fast plate processing time of 1 minute per plate, it is perfectly suited for screening applications with maximum reproducibility. Unfortunately, no single delivery method or transfection reagent can be applied to all types of cells. Product overview the 384well nucleofector tm system is an independent platform for highthroughput nucleofection in a 384well format. Transfection efficiency and survival rate achieved. In practical terms, reverse transfection refers to cell based array experiments in which cells are pipetted onto preplated cargo molecules.
During the optimization process, a range of conditions for different transfection. Aug 31, 20 the very basic requirement for the existence of genetic engineering is a simple fact. Using neon pipette and tip, transfect 10ul of cell rna mixture. Many cell biology experiments require the introduction of genetic material into cells. Electroporation parameters for dna plasmids 15kb using. Fortebio octet qk labelfree analyzer with computer and octet software lot 400. 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. Neon transfection system feb232010 hello, we think of acquiring soon a new transfection system from invitrogen, the neon. Cell type specificity and structural determinants of ires activity from. Protocol for electroporation of cas12a ribonucleoprotein rnp into. Starrlab electroporation using neon electroporation from.
Monitor the progress of the transfection every few days by observing the amount of cell death and the color of the culture medium. I followed the manufacturers online protocol for 3t3l1 cells. Reagent agent transfection database from the transfection experts. Brightfield and fluorescence microscopy images of the cells following electroporation. Use this selection table to find the neon transfection system electroporation conditions, transfection efficiency and survival rate and microscopy images of cells. Neon transfection system thermo fisher scientific es.
Neon electroporation transfection neon transfection system cell line data the table below contains a listing of the cell lines and primary cells successfully electroporated with the neon transfection system or the predecessor instrument the microporator mp100. Jackie webb scientist i, thermo fisher scientific speaker. The neon transfection system enables fast and efficient delivery of nucleic acids. Using neon pipette and tip, transfect 10ul of cell. Nk92 cell line, freshly isolated resting or il2activated nk cells were electroporated with the neon transfection system thermo fisher scientific, waltham, massachusetts, usa. Transfection cell database our searchable database contains data from researchers like yourself who have shared their experimental results with us. With the introduction of new systems for transfection such as the neon transfection system. Transfected hek293 cells were collected, washed once in pbs, and lysed. Optimize dna transfection transfection, electroporation. Both systems can result in the efficient transfection of hesc. The development of reporter gene systems and selection methods for stable maintenance. Thermofisher neon transfection system 10 l kit mpk1025. Transfection is a powerful analytical tool enabling study of the function of genes and gene products in cells. Request a sample for low toxicity transfection mirus bio.
Universal transfection reagent protocol sigmaaldrich. Typically, 24 to 48 hours between transfection and activity assay is sufficient. The hyperlinked cell line names take you to a pdf that gives. When working with a new cell type, test a range of reagent volumes at a fixed dna amount to find the level that achieves the highest transfection efficiency with minimal toxicity. Transfection is the process of inserting genetic material, such as dna and double stranded rna, into mammalian cells. The basic, single cuvette based nucleofector tm system for efficient transfection of hardtotransfect cell lines and primary cells with different substrates e. Cells were transfected using the neon electroporation system. Optimization of gene transfection in murine myeloma cell. The underlying reason is that this method, which employs electrical pulses to create transient pores in cell membranes, reproducibly yields high transfection. Efficient, highthroughput transfection of human embryonic. Flow cytometric data were analyzed using the program, expo32adc. Has anybody done transfection of b lymphocytes using neon.
Gibco 24 hours of stem cells virtual event presented by. Neon transfection system cell line data thermo fisher. Btw for b cell electroporation, including b cell in the pbmc fraction or cd40l expanded b cells, electroporation works best with celetrix electroporation system at about 80% efficiency, much. Optimize dna transfection transfection, electroporation and. As a bit of background, transfection is a method to transfer genetic material into cells in order to add or alter the expression of a specific gene in a cell. Invitrogen neon transfection system mpk5000 electroporation. Apr 30, 2019 nk92 cell line, freshly isolated resting or il2activated nk cells were electroporated with the neon transfection system thermo fisher scientific, waltham, massachusetts, usa. The manufacturer typically has a database with a recommended setting for a given cell. With an extremely fast plate processing time of 1 minute per plate. Pc12 is an established rat pheochromocytoma cell line, which responds to exposure to ngf with cessation of growth, expression of cytoplasmic processes, and differentiation into cells resembling sympathetic. Transfection the delivery of dna or rna into eukaryotic cells is a powerful tool used to study and control gene expression. Very important to make sure that there are no bubbles in the tip. A variety of viral and nonviral delivery systems have been developed, but electroporation is often the selected method of choice.
Get highly efficient transfection of primary cells, stem cells, and difficultto transfect cells with the versatile and easytouse neon transfection system. Transfection guide overview of transfection methods promega. Get improved transfection outcomes with the neon transfection system. Remove the pipette from the station and gently expel the cell dna mixture by. Transfection of mammalian cells with fluorescent protein fusions. During the optimization process, a range of conditions for different transfection parameters dna concentration, number of cells, pulse settings was tested. Transfection experiments are conducted in order to study gene expression, cell.
Invitrogen neon transfection system 100 l kit 96 x 2. Invitrogen neon transfection system starter pack 1 pack. Mouse myeloma cell lines sp20, ns0, ns1, ag8, and p3u1 were transfected with pegfpn1 vector using lipofectamine 2000, jetpei and lyovec commercial transfection reagents in different combinations. Transfection is the process of introducing nucleic acids into eukaryotic cells by nonviral methods. The flexible and open system allows the user to perform highquality transfections using optimized or userdefined protocols in three simple steps with as few as 2 x 10 4 cells. Some of the transfection efficiency and viability data was generated with the microporator mp100, the predecessor or the neon transfection system see. Electroporation parameters for dna plasmids 15kb using the. The very basic requirement for the existence of genetic engineering is a simple fact. Jul 12, 20 a wide variety of mammalian cell types is used in gene transfection studies. Rat dp stem cells have been successfully transfected using calcium phosphate. Transfection methods and techniques altogen biosystems. Established cell lines, hardtotransfect cells, primary cells, stem cells. Little is known about transfectability of murine myeloma cell lines by different transfection reagents.
Invitrogen neon transfection system starter pack life. Established cell lines, stem cells, primary cells, hardtotransfect cells. Ncbi gene expression omnibus database under accession number. General information about the cell line or primary cell. As always, qiagen cannot verify data supplied from outside sources. The insertion of dna into a cell enables the expression, or production, of proteins using the cells own machinery, whereas insertion of rna into a cell. The neon system employs specialized consumable pipette tips containing goldplated electrodes as electroporation chamber.
Jun, 2010 transfection is a powerful analytical tool enabling study of the function of genes and gene products in cells. The neon transfection system 10 l kit is designed specifically for use with the. For detailed instructions, refer to the manual supplied with the product or download the manual from. I followed the manufacturers online protocol for 3t3l1 cells 1,300v, 20 ms, 2 pulses. Press the pushbutton on the neon pipette down to the first stop, immerse in the celldna mixture and slowly release the pushbutton to suck up 100 l of celldna mixture. Reagent agent transfection database from the transfection. Altogen cro offers in vivo rnai services, tumor xenograft models, toxicology testing, stable cell. Animal originfree, serumfree and designed specifically for use with the neon transfection system. At this point i have a set of tubes with cells and appropriate rna combined.
Msms spectra were searched against the crap protein sequence database. The common causes of transfection failure are decrease transfection efficiency and decreased cell viability. A free program designed to help you achieve your scientific aspirations. Some cells, like hela cells, are easy to transfect with lipid reagents. Plate cells to obtain 7080% cell density on the day of transfection. The insertion of dna into a cell enables the expression, or production, of proteins using the cells own machinery, whereas insertion of rna into a cell is used to downregulate the production of a specific protein by stopping translation. Request a free sample of low toxicity transfection reagents. L, containing 5 x 10 42 x 10 5 adherent cells or 1 x 10 65 x 10 5 suspension cells. All available information is shown in the detailed view. Intracellular uptake of reporter vector encoded with egfp at 24 hr following transfection of jurkat cells with neon transfection system. A costeffective approach to microporate mammalian cells. In general, the use of such a capillary electroporation system which is often called a microporation device has important advantages compared with conventional cuvettebased electroporation chambers.
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. While the neon system had a higher transfection efficiency, it also had increased variability between multiple transfections 36. An electroporation protocol for efficient dna transfection in. For use with the neon transfection system use this kit for transfection volumes of 100l, containing 5 x 10 5 2 x 10 6 adherent cells or 1 x 10 6 5 x 10 6 suspension cells cells that have been transfected using the included 100l neon tips are then ready to be washed and plated into a 6well, 60mm, or 10cm culture dish, depending on protocol. Search for publications in the transfection database with polyplus transfection reagents or for transfection conditions. Transfection of mammalian cells with fluorescent protein. Sigmas universal transfection reagent is a unique formulation of a proprietary polymer blend used for transient and stable transfection of nucleic acids into various eukaryotic cell lines and hardto. Jun 29, 2010 the optimum ratios of dna and transfection reagents obtained for a number of cell lines, including two of the myeloma cell lines sp20 and ag8 were found to be 2 g 4 l, 2 g 2l and 0. Neon transfection system thermo fisher scientific us. An electroporation protocol for efficient dna transfection. The optimal time period depends on the protein being expressed, and thus the sensitivity of the assay. Our universal transfection reagent is a unique formulation of a proprietary polymer blend.
Transfection efficiency testing using fluorescenceactivated cell sorting. A costeffective approach to microporate mammalian cells with. For detailed instructions, refer to the manual supplied with the. Efficient electroporation with neon transfection system. Cells that have been transfected using the included 10 l neon tips are rea. A wide variety of mammalian cell types is used in gene transfection studies. Any gaps between the pipette tip and the pipette will cause poor transfection. Neon transfection system cell line data thermo fisher scientific. Cloned genes can be transfected into cells for biochemical characterization, mutational analyses, investigation of the effects of gene expression on cell. Neon transfection system protocols and cell line data thermo. Highefficiency genomic editing in epsteinbarr virustransformed. High transfection efficiency of jurkat cells with life technologies neon tranfection system.
Transfection guide overview of transfection methods. The neon transfection system uses a single transfection kit neon kit that is compatible with various mammalian cell types including primary and stem cells thereby avoiding the need to determine an optimal buffer for each cell type. Using various chemical or physical methods, this gene transfer technology enables the study of gene function and protein expression in a cellular environment. All mass spectra from were analyzed with maxquant software version. For large plasmids, it is important to prepare a highly concentrated solution of the plasmid for electroporation. Fast and easy protocol is compatible with serum, serumfree medium and antibiotics.
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