Transfections can be carried out entirely in the presence of serum. While the application of bacterial transformation is typically for overexpression of genes in an effort to produce large quantities of proteins that can be purified and studied, dna transfection is typically used for the studying the effect of gene expression in a cellular context. Manufacturer specifications amaxa 4dnucleofector system, lonza please note. Transfection protocols often require serumfree conditions for optimal performance because serum can interfere with many commercially available transfection reagents. Cell transfection device transfecting hardtotransfect cells e. The nucleofector ii2b transfection device is the single cuvette based system that has been used in research labs since 2001, and formerly known as the amaxa device. The neon transfection system is a benchtop electroporation device that uses the pipette tip as an electroporation chamber to efficiently transfect mammalian cells including primary cells and stem. It allows efficient transfection of hardtotransfect cell lines and primary cells for transfection in lowthroughput format with. Dna transfection is the process by which dna is taken up by eukaryotic cells.
Nucleofection is a technology based on the momentary creation of small pores in cell membranes by applying an. Use ingenio with amaxa nucleofector device ii system for hardtotransfect cell types. Transfecting human neural stem cells with the amaxa nucleofector. A nucleofectortm device that comprises unique electrical parameters pre programmed for each optimized cell type, to deliver the substrate directly into the cell. Transfecting neural cells using the neon transfection system. Transfection is generally defined as the process of introducing dna, rna or proteins into cells to influence their genotype or phenotype. 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. Does anyone have experience with amaxa nucleofector. The cuy21edit electroporator is desigend for in vivo, in ovo, in utero and ex vivo electroporation applications. Amaxa transfection device nucleofection is a technology based on the momentary creation of small pores in cell membranes by applying an electrical pulse.
Xfect is a transfection reagent that creates biodegradable nanoparticles that permit superior transfection efficiency of plasmid dna into mammalian cells. Trouble with amaxa nucleofection in my lab we need to deliver dna to b cells. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. Only use the device when it is set on top of a safe, plain and stable table or bench. Do not plug any nonamaxacertified connectors into the external electrode sockets at. This protocol is modified from the manual provided by the kit and device manufacturer lonza cologne ag. Plasmid dna transfection of ct26 mus musculus colon. Transfection reagents are highly efficient for dna and sirna transfection in vivo and in vitro. Altogen biosystems provides in vivo transfection reagents, over 100 preoptimized in vitro transfection kits for cell lines and primary cells, and electroporation delivery products. The nucleofector device is essentially a cell electroporator that is programmed with various profiles that are defined electrical parameters for specific cell types.
Nucleofector device and cell specific nucleofector kits. Our products speak for themselves in quality and performance. Transfection of bloodstream and procyclic trypanosomes amaxa transfection of bloodstream forms if you transfect bloodstream forms the amaxa nucleofector ii gave us up to follow the links for test results as pdf a 300fold increase in transient transfection, as judged by luciferase expression, and about 70fold increase in stable clones. Protocols and techniques vary widely and include lipid transfection and chemical and physical methods such as electroporation. Using amaxas 96well shuttle, a high throughput extension for the well established nucleofector platform, transfection. The optimization strategy is suitable both for optimizing nucleofection of plasmid dna, as well as, sirna oligonucleotides. Transfection definition of transfection by medical. Using this protocol, we have achieved initial transfection efficiencies of 35% and 70% after stable transfection. Spin down cells 3000rpm, 10 min, rt remove supernatant by sterile aspiration. The common causes of transfection failure are decrease transfection efficiency and decreased cell viability. Transfection protocols vary based on cell type to be transfected, transfection method, and transfection reagent. Insert screw driver supplied with the 4dnucleofector system in the lower hole of. Please note that the amaxa nucleofector technology is not intended to be used for diagnostic purposes or for testing or treatment in humans.
May 25, 2007 transfection is an essential tool for numerous applications, including studies of gene expression. Accordingly, the cuy21sc is primarily characterised by its finer output voltage control. Transfection system the neon transfection system is a novel, benchtop electroporation device that employs an electroporation technology by using the pipette tip as an electroporation chamber to efficiently transfect mammalian cells including primary and immortalized hematopoietic cells, stem cells, and primary cells. This protocol is modified from the manual provided by the kit. High efficiency electroporation using any instrument. Transfecting human neural stem cells with the amaxa. Using the amaxa minipipette, transfer the celldna mix into the amaxa transfection cuvette. Growth mia paca2 cells in t75 cell culture flasks as described above.
Does anyone have experience with amaxa nucleofector based. Nucleofector ii from amaxa biosystems biocompare product. Do not obstruct the movement of the carousel of the nucleofector. However, transfection of cells with common methods such as calcium phosphate, deaedextran, liposomemediated transfer, electroporation or viral vectors is limited because of low transfection efficiency and the extended time required for analysis. Use the table below to troubleshoot transfection experimental failure. Hek293, hela, thp1, jurakat e61, vero, nih3t3, mcf7, huvec. The optimization strategy is suitable both for optimizing nucleofection of plasmid dna, as well as. To view an uptodate list of all cell lines for which either an optimized protocol or customer data.
Altogen cro offers in vivo rnai services, tumor xenograft models, toxicology testing, stable cell line generation, and. Chos cells were transfected with an igg expression plasmid via small or largescale electroporation using the maxcyte stx. Wash a defined number of cells with pbs we usually use 5 x 106 cells per transfection. They are not compatible with the nucleofector ii2b device. What has been our mission in the past will continue to be so in the future providing you with innovative transfection solutions to help you thrive and excel in your research. Transfection of cells with a protein of interest the following protocol describes the transfection of protein into one well of a 6well plate. The neon transfection system is a benchtop electroporation device that uses the pipette tip as an electroporation chamber to efficiently transfect mammalian cells including primary cells and stem cells. Allow pellet to settle and remove hbss and parts of the pellet so that you will have a final volume of about 200.
It enables highly efficient, transfection of primary cells, stem cells, neurons, and cell lines that have traditionally been difficult to transfect via electroporation and other nonviral. It was characterised by a large extended outputvoltage range. Stable transfection viafect transfection reagent can be used to produce stable transfectants. Allow pellet to settle and remove hbss and parts of the pellet so that you will have.
Manufacturer specifications amaxa 4dnucleofector system. The comprehensive way in which nucleofector programs and cell typespecific solutions are developed enables nucleic acid substrates delivery not only to the cytoplasm, but also through. Manufacturer specifications amaxa 4dnucleofector system, lonza. Today, efficient and reproducible high throughput transfection remains a challenge. Instructions for using the neon transfection system for transfecting of neural cells are described below. The nucleofector technology, comprising nucleofection process, nucleofector device, nucleofector solutions, nucleofec. Therefore, use this device with care and take the following precautions. Ingenio electroporation products for high efficiency electroporation. All i know from colleagues using amaxa is that its a very efficient for transfection of cell lines that other methods cant efficiently. This device is designed to deliver variable high voltage electrical impulses for the purpose of introducing nucleic acids into eukaryotic and prokaryotic cells.
It allows efficient transfection of hardto transfect. Transfection definition of transfection by medical dictionary. Products mirus bio transit certified transfection reagents. I want to transfect murine hematopoietic progenitors with a cas9encoding. Make sure you have 1 ml of prewarmed culture media for the next step.
Lab products cell biology cell transfection magnefectnano ii. However, we recommend optimizing transfection conditions using transient transfection studies prior to selecting for stable transfectants. Amaxa 4dnucleofector optimization protocol for cell lines. Biopharmaceuticals are one of the key growth drivers of the.
Also, remove the cell dish with culture media from the incubator and place it in the tissue culture hood. All i know from colleagues using amaxa is that its a very efficient for transfection of cell lines that other methods cant efficiently tranfect and primary cells. The amaxa nucleofector technology is a transfection system designed to transfer genes into cells quickly and efficiently. Automated transient transfection for high throughput. It may also refer to other methods and cell types, although other terms are often preferred. Protocols and techniques vary widely and include lipid transfection and chemical and physical. Comparison of transfection efficiency between electropo. At mirus bio, our role in programming the genome is to provide scientists turnkey tools to add, delete, or modify any gene at will. Before choosing a transfection method, it is important to consider what substrate to transfect and whether you wish to achieve transient or stable transfection. Expertise in cellular and molecular biology is behind the invitrogen product portfolio of dna reagents and consumables, which are the most cited and trusted. The next few steps have to take less than 15 min, so do them. Xtremegene transfection reagents from roche have been used for a wide range of applications and a vast array of cell lines, primary cells and stem cells.
Our solution is an improved electroporation technology, the nucleofector tm technology, originally introduced into the market by legacy amaxa in 2001. None of the equipment posted for sale on is owned by medwow, should you have any questions regarding a specific item, please direct them to the appropriate seller by making use of the available communication channels on the items page. The technique is proprietary, so its hard to tell exactly whats going on. The lonza 4dnucleofector system white bar resulted in significantly higher transfection rates compared to all. Cell transfection device integrated scientific solutions inc. Protein expression from plasmid dna transfected into cultured cells is a powerful tool to. Ar42j transfection reagent pancreatic carcinoma altogen. They are resistant to easy transfection methods and lately we have been having issues with our outdated retroviral system. Transfection methods reagentbased methods deaedextran method overview solution a. Transfection is the process of deliberately introducing naked or purified nucleic acids into eukaryotic cells. Pellet the cells by centrifugation rpm 200xg for 5 minutes, remove as much pbs as possible, and resuspend the cell pellet in 100 l of the. Further development of electroporation by legacy amaxa has led to the development of a more efficient transfection technology called nucleofection. Neurons were identified by betaiiitubulin labeling upon fixation after 48 h in culture.
Ezporator electroporation system and lonzaamaxa nucleofector ii2b devices 0. We off er 3 diff erent device platforms plus an addon device. They are designed to connect amaxa certified addon devices to the. Using amaxas 96well shuttle, a high throughput extension for the well established nucleofector platform, transfection of difficulttotransfect cell lines and primary cells is no longer out of reach. Electroporation devices cuy21 edit sc sonidel limited. Amaxa transfection device american university of beirut. Following trituration of cortices, carefully wash the pellet in 5ml hbss. The electrical settings are preprogrammed for each optimized cell type and can be selected via the device or a pc software. Transfection transfection is the process by which nucleic acids are introduced into mammalian cells. We describe here a stepbystep protocol for transfecting hnspcs using amaxa s nucleofector device and technology with electrical current parameters and buffer solutions specifically optimized for transfecting neural stem cells. See table 1 at the end of this section for information on.
Transfection is an essential tool for numerous applications, including studies of gene expression. While the application of bacterial transformation is typically for overexpression of genes in an effort to produce large quantities. Further development of electroporation by legacy amaxa has. Here i describe the general protocol for using the amaxa nucleofector device and kit to deliver sirna, using mia paca2 cells as an example.
None of the equipment posted for sale on is owned by medwow, should you have any questions regarding a. Amaxa sirna feb2009 i am using amaxa kit to knock down a transcription factor expression in sknsh cells, but got offtarget effect do you know anyway to fix this. The nucleofector device the nucleofector device delivers unique electrical parameters. The nucleofectortm device is the single cuvette based system that has been used in research labs since 2001. We describe here a stepbystep protocol for transfecting hnspcs using amaxas nucleofector device and technology with electrical current parameters and buffer solutions specifically optimized for. However, transfection of cells with common methods such as calcium phosphate, deae. To implement high throughput transient transfection, each step of sample preparation leading to the automated transient transfection process needs to be planned and integrated. Does anyone have experience with amaxa nucleofector based transfection of murine hematopoietic progenitor cells. Place the dnas to be transfected, and transfection solution from amaxa, on ice. Use ingenio with amaxa nucleofector device transfection. Place the device such that easy removal of the power cord is possible at any time. Nucleofector device and cell specifi c nucleofector kits. Unfortunately, no single delivery method or transfection reagent can be applied to all types of cells. A single cuvette based system used for the transfection of cell lines and primary cell that are hard to infect with various substrates including dna vectors or sirna olignucleotides.
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