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