The neon transfection device with its integrated buffer kit works with cell lines, primary cells, and stem cells. High efficiency transfection of icell cardiomyocytes and stem. Invitrogen neon transfection system 100 l kit 96 x 2. 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 neurons.
Get improved transfection outcomes with the neon transfection system. Transient plasmid dna transfection into hela and hacat cells. 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. As for all transfection methods, electroporation has its advantages and disadvantages. Optimization of squarewave electroporation for transfection. 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.
Input the electroporation parameters in the input window, if you already have the electroporation parameters for your cell type. The electroporation method we describe is designed for the neon transfection system, but is adaptable to other systems. Targeted mutagenesis in wheat microspores using crispr. Efficiencyup to 90% transfection and geneediting efficiency in extremely difficult cells, including immune, primary and stem cells. Enter electroporation settings, or choose setting from the optimization protocol.
Mpk1025, mpk1096 optional geneart genomic cleavage detection kit. 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. Successful crispr genome editing in hardtotransfect. Several years ago, a new flexible and easy to operate electroporation device was launched under the name neontm transfection system. Nakayama a, sato m, shinohara m, matsubara s, yokomine t, akasaka e, yoshida m, takao s. The neon 10 l transfection system draws 10 l of cells and transfection material into an electroporation pipette tip. This device allows you to transfection directly in the pipette tip, limiting the steps required. Quick howto video on how to use and conduct the optimization protocols for the neon transfection system for electroporation instrument. However, the relatively high costs of electroporation kits and reagents temper the routine use of this fast and easy to perform technique in many laboratories. In this article we describe the optimization of transfection with transfectin monitored by a secreted alkaline phosphatase seap reporter gene assay. Set up the neon pipette station by filling the neon tube with electrolytic. High efficiency transfection of icell cardiomyocytes and. Unpacking the neon transfection system unpacking instructions follow the instructions below to unpack the neon transfection system.
In animal cells, transfection is the preferred term as. Sigmaaldrich developed its nter nanoparticle sirna transfection system. The ingenio electroporation kit was used to transfect 2 x 10 6 ips cells on the amaxa nucleofector ii device b. The neon transfection system is a novel, benchtop electroporation device that employs an electroporation technology by using the pipette tip. Cells that have integrated foreign dna in their genome. 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. Morgan surgery branch, national cancer institute, national institutes of health, bethesda, md 20892, usa. Efficient transfection of primarily cultured porcine embryonic fibroblasts using the amaxa nucleofection system. Crisprcas9mediated genome editing of human primary tcells.
Im using life techs neon transfection kit to transfect primary adrenal medullary cells with various constructs for overexpression. In summary, the optimised protocol for wheat microspore transfection using the neon system involves electroporation of a minimum of 75000 cells in. Neon transfection system in vivo delivery invivofectamine 2. Efficient, highthroughput transfection of human embryonic. Successful crispr genome editing in hardtotransfect cells.
Corroborating the efficacy of k2 transfection system, we obtained satisfying results also by transfecting cells for only 6 hours instead of the 24 hourlong incubation of cells with the dnak2 transfection mixture, as recommended by the manufacturer not shown. An electroporation protocol for efficient dna transfection. A costeffective approach to microporate mammalian cells. Concentrated antibody targeting your protein of interest 0. Invitrogen instructions say to use buffer e if electroporating in 10 l and e2 if electroporating in 100 l. In our eperiments, the optimum settings for urkat cells was found to e 1600 v, 10 ms pulse width, 3 pulses 2. Plasmid dna transfection of ct26 mus musculus colon carcinoma. Using a 10l neon transfection system tip, we followed the neon optimization protocol publication number man0001557, revision a. Transfection efficiency was measured by flow cytometry for the expression of the green fluorescent protein and the viability of the transfected cells was determined by an atp catalyzed luciferase reaction.
Cells that have incorporated the foreign dna are called transfectants. It may also refer to other methods and cell types, although other terms are often preferred. Yasuhiko hayakawa with over 20 years of experience in the field of electroporation and cell fusion, he founded nepa gene company. Ingenio ezporator pulse generator system electroporation device. Enter electroporation settings, or choose a setting from the optimization protocol. Reduction of thiolmodified oligonucleotides 575 kb reduction of thiol. In our experiments, the optimum settings for jurkat cells was found to be 1600 v, 10 ms pulse width, 3 pulses 3. For use at room temperature under normal laboratory conditions. Transient transfection of cells and evaluation of transfection efficiency for optimization of transfection conditions, 90. A popular electroporation device is thermofisher scientifics neon transfection system. The hyperlinked cell line names take you to a pdf that gives. Several years ago, a new flexible and easy to operate electroporation device was launched under the name neon tm transfection system. Crisprcas9mediated genome editing of human primary t. Highefficiency transfection of primary human and mouse t lymphocytes using rna electroporation yangbing zhao, zhili zheng, cyrille j.
Guideit recombinant cas9 electroporationready user. Using the neon transfection system refer to the manual for details on setting up the neon device and neon pipette station. On the plus side, you can use it to transfect large dna fragments and achieve good transfection efficiencies using cell lines. Therefore, the volumes in this protocol are for duplicate reactions set up in the same tube, with an overage of 5 l.
Guideit recombinant cas9 electroporationready user manual. To establish a method to introduce mrna into human fibroblast cells. Apr, 2011 the neon transfection system is a next generation electroporator with some unique benefits. A benefit of this device is that it is an open system and the user can manually set the parameters for each sample. Avoid creating bubbles, which can hinder electroporation. Panel b illustrates camp induction measured via a luciferase reporter plasmid. Transfection is the process of deliberately introducing naked or purified nucleic acids into eukaryotic cells. A costeffective approach to microporate mammalian cells with the neon. Has anybody done transfection of b lymphocytes using neon. Please refer to the neon transfection system user manual and manufacturers website for detailed operating instructions for the neon transfection system.
At this point i have a set of tubes with cells and appropriate rna combined. From time to time, when using the neon transfection system, i do not see the side electrode pulsing, which i would expect to see sometime between. This system uses an electronic pipette tip as a transfection chamber instead of the standard electroporation cuvette, which makes it more flexible for variations in cultures. This system is designed to be used with the ingenio electroporation solution which is ideal for mammalian and insect cell transfection. Life tech neon transfection system solution r approximation. Efficient transfection of human fibroblast cells with mrna by microporation. 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.
Neon transfection system 862 kb altr crisprcas9 systemrnp electroporation, neon transfection system. Cells were transfected with k2 transfection system biontex. This characteristic allows one to optimize specific parameters for each cell type. Using neon pipette and tip, transfect 10ul of cellrna mixture. Neon transfection of primary mouse t cells the ansel lab. Forthe electroporation with theneonsystem,pc12 cells were grown to 70 % con. Set up the neon pipette station by filling the neon tube with. The neon transfection system enables fast and efficient delivery of nucleic acids into all mammalian cell types, including primary, stem, and difficulttotransfect cells. Establishing transfection methods that enable highly efficient dna uptake has become increasingly important. Generate induced pluripotent stem cells ipscs transfect stem cells day 14 day 17 bj hdfn hdfa lipofectamine 3000 reagent neon system electroporation a b dna. Protocol for electroporation of cas12a ribonucleoprotein. An electroporation protocol for efficient dna transfection in. Isolate the tcells from peripheral blood mononuclear cells pbmc derived from healthy donors using the dynabeads 10 untouched human t cells kit. Jackie webb scientist i, thermo fisher scientific speaker.
A costeffective approach to microporate mammalian cells with. Ingenio ezporator pulse generator system electroporation. 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. Thermo invitrogen neon mpk5000 transfection system youtube. Cell density, duration of transfection, volume of medium during transfection, and ratio of lipid reagent to dna are key factors for efficiency. While these protocols may serve as a helpful starting point for electroporation of other cell types as well, further optimization will be required.
Efficient electroporation with neon transfection system. Dec 30, 2010 efficient transfection of human fibroblast cells with mrna by microporation. Set the desired pulse parameters on the neon machine. Techniques vary widely and include lipidbased transfection and physical methods such as. 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. Techniques vary widely and include lipidbased transfection and physical methods such as electroporation.
Neon transfection system in vivo delivery invivofectamine3. Protocol for electroporation of cas12a ribonucleoprotein rnp. Thermos neon transfection system issue researchgate. Fill plastic neon tube with 3 ml of invitrogen electrolyte buffer and insert tube into the neon pipette station.
Cells were analyzed with the attune nxt flow cytometer 24 hours post transfection. Neon transfection system cell line data thermo fisher. The neon system employs specialized consumable pipette tips containing goldplated electrodes as electroporation chamber. Introduction of foreign dna into the nucleus of eukaryotic cells. The neon transfection system enables fast and efficient delivery of nucleic acids into all mammalian cell types, including primary, stem, and difficulttotransfect. The neon transfection system offers open and transparent protocols that are optimized for ease of use. Neon transfection system enables fast and efficient delivery of nucleic acids into all mammalian cell types, including primary, stem, and difficulttotransfect cells.
The neon transfection system comes with a neon electroporation device, pipette, pipette station, usb thumbdrive, manual and power cord. I followed the manufacturers online protocol for 3t3l1 cells 1,300v, 20 ms, 2 pulses. Save the outer box and other packaging material in case you need to transport or ship the unit. Invitrogen neon transfection system starter pack 1 pack. The 24well optimization protocols were performed using the 10 l neon tip, and the cells were dispensed into 0. Neon transfection system 862 kb altr crisprcas9 systemrnp electroporation, neon transfection. Transfection by electroporation dna electroporation was performed with the neon transfection system mpk5000 invitrogen or the gene pulser xcell system biorad, hercules, ca, usa. General information about the cell line or primary cell. Tutorials and ipython notebooks to get users started with using neon for deep learning. Highefficiency transfection of primary human and mouse t. Efficiency of gene transfection into donor cells for nuclear transfer of bovine embryos. Ive found that while they suggest tossing electroporation tips after two uses i can easily go up to. There are 3 options to select an electroporation protocol for your cell type.
Electroporation is the method of choice for many hardtotransfect cell types, and the ingenio ezporator electroporation system is a costeffective, straightforward, open system that is perfect for any lab seeking performance without breaking the bank. Electroporated cells 10 l were mixed with 175 l media, and 50 l diluted cells were plated in. Instructions for using the neon transfection system for transfection of mammalian cells are described below. However, transfection efficiency in primary cells is low, mostly due to the high mortality rates caused by the electric pulses. Jun 14, 2018 thermo invitrogen neon mpk5000 transfection system is available from the lab world group. Gibco 24 hours of stem cells virtual event presented by. Lipofectamine 3000 reagent or the invitrogen neon transfection system. Pdf an electroporation protocol for efficient dna transfection in. To establish a method to introduce mrna into human fibroblast cells, we utilized the neon transfection system invitrogen. The protocol below assumes the use of a neon transfection system and accessories buffers, etc. Electroporation using neon electroporation from invitrogen.
L of the tcells mixed with cas9grna complexes into the neon. Transient plasmid dna transfection into hela and hacat. Intracellular uptake of reporter vector encoded with egfp at 24 hr following transfection of jurkat cells with neon transfection system. Thermo invitrogen neon mpk5000 transfection system is available from the lab world group. Neon transfection system thermo fisher scientific us. The optimal amount of dna to use in the transfection will vary widely depending upon the type of dna, transfection reagentmethod, target cell line, and number of cells genetic material dna quality and quantity factors affecting transfection 8. This tip may be used twice for two sequential electroporations.
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