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amaxa eNews #4
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Latest Advances in Nucleofector Technology for the Transfection of Primary Neurons
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Whether you are performing biomolecular analyses such as quantitative PCR or Western Blot studies, or analyzing your samples via microscopy, Nucleofector Technology is the ideal tool to advance your neuroscience research.
amaxa’s latest products facilitate high-efficiency nucleofection of primary neurons using very small cell numbers, in single cuvette or in microplate format.
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NEW: The Basic Neuron Small Cell Number Nucleofector Kit
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High-efficiency transfection of low-abundant neurons in single-cuvette format
amaxa’s new Basic Neuron Small Cell Number (SCN) Nucleofector Kit allows high efficiency transfection even for extremely low cell numbers. Now you can nucleofect only the number of cells you need for later culture and analysis (e.g. microscopic analyses of neuron growth and morphology).
Benefit from a drastic reduction in both number of donor animals and amount of preparation time. The kit includes new, optimized SCN Nucleofector Solution, SCN cuvettes and programs which enable transfection efficiencies of up to 60% with as little as 15,000 cells per nucleofection. The kit has been successfully tested for many low-abundant primary neurons such as DRG (figure 3) and hippocampal neurons from mouse and rat.
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Small Cell Number Nucleofection – Benefits at a Glance
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| Transfection of as little as 15,000 cells per nucleofection | More experiments with fewer donor animals
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| | Excellent non-viral transfection efficiencies and viabilities | 50% transfection efficiency and higher, even for very difficult-to-transfect neurons like DRGs
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| | Suitable for broad range of low-abundant primary neurons | Transfection of your specific type of primary neuron is supported by a panel of nucleofection programs and a growing number of Optimized Protocols with individual culture details.
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Figure 1: SCN nucleofection of embryonal rat hippocampal neurons.
20,000 freshly isolated rat hippocampal neurons (E17) were nucleofected with with SCN Basic Neuro Program 1, 0.4 μg pmaxGFP and seeded on a coated coverslip. 24 hours post-nucleofection, neurons were fixed and analyzed by light (A) and fluorescence (B) microscopy. Transfection efficiency with optimized conditions ranged between 40 - 65%, depending on preparation. Neuron morphology was unaltered compared to untransfected neurons after 24 hours and 7 days (data not shown). Data courtesy of M. Kiebler, Department of Neuronal Cell Biology, Medical University of Vienna, Vienna, Austria.
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Figure 2: Average transfection efficiencies and viabilities of embryonal rat hippocampal neurons (n = 3). 20,000 and 50,000 cells were nucleofected, cultured and analyzed as described above. Viability was determined as number of living cells compared to non-nucleofected control. Data courtesy of M. Kiebler, Department of Neuronal Cell Biology, Medical University of Vienna, Vienna, Austria.
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Figure 3: SCN Nucleofection of freshly isolated mouse Dorsal Root Ganglion (DRG) cells. 100,000 (A) and 15,000 (B) DRGs were nucleofected with SCN Basic Neuro Program 6 and 0.4 μg pmaxGFP (green) and seeded on a coated coverslip. 24 hours post nucleofection, cells were fixed DAPI-labelled (figure A, blue, nuclei) and immunostained for beta-tubulin (red, neuronal marker). A merge of all is shown in figure on the right side. Transfection efficiency as determined by fluorescence microscopy was approximately 60% in both experiments. Data courtesy of B. Eickholt, MRC Centre for Developmental Neurobiology, King‘s College, London, United Kingdom.
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High-throughput Transfection of Primary Neurons
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amaxa’s Nucleofector 96-well Shuttle System opens a new dimension in neurobiology research. siRNA and shRNA library screens, as well as other applications demanding high-throughput transfections, are no longer out of reach for neurobiologists. Due to the low nucleofection volumes, considerably few cells are required per transfection (down to 50,000 cells in 20 μl). The use of Nucleocuvette multiwell modules (16-well modules which can be combined to a Nucleocuvette 96-well plate), offers additional advantages in nucleofection of primary neurons.
The microplate format significantly decreases the hands-on time required for nucleofection of multiple samples compared to a single-cuvette system. This, in combination with the short processing time, allows the 96-well Shuttle to drastically reduce both time and cell material required per experiment. This greatly increases the number of experiments which can be realized with a single preparation of primary neurons, as well as overall throughput.
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High throughput Nucleofection of Primary Neurons – Benefits at a Glance
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| High throughput transfection using primary neurons | siRNA and shRNA library screens are made possible for primary neurons
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| | Excellent non-viral transfection efficiencies and viabilities | 50% transfection efficiency and higher even for very difficult-to-transfect neurons like DRGs
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| | Optimized for most common primary neurons | Optimized Protocols for nucleofection of hippocampal neurons, cortical neurons, DRGs and many more, including comprehensive guidance for cell culture.
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Figure 4: Normal differentiation and morphology of neurons after 96-well nucleofection. Especially for neurons, which are very susceptible to external stress, it is crucial that the transfection procedure does not have any artificial side-effects on morphology and differentiation of neurons. P0 neurons from rat hippocampus were nucleofected with pSyn-GFP and pDsRed and plated onto glass cover slips and examined 7 days in-vitro for GFP (green) and RFP (red) expression. Transfected neurons show an extensive dendritic network and, as shown in the magnification, develop dendritic protrusions that already resemble mature, mushroom-shaped dendritic spines (arrows). Surrounding, untransfected glia cells are shown by DAPI staining (blue). Data courtesy of M. Kiebler, Department of Neuronal Cell Biology, Medical University of Vienna, Vienna, Austria.
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Ordering information
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| Cat.No. | Devices | | AAD-1001 | Nucleofector II Device | | AAM-1001 | 96-well Shuttle | | | | | | Nucleofector Kits for Primary Cells | | NEW! VSPI-1003 | Basic Neuron SCN Nucleofector Kit* | | VPG-1006 | Astrocytes, mouse | | VPG-1007 | Astrocytes, rat | | VPG-1004 | Neural Stem Cells (NSC), mouse | | VPG-1005 | Neural Stem Cells (NSC), rat | | VPG-1002 | Neurons, chicken | | VPG-1001 | Neurons, mouse | | VPG-1003 | Neurons, rat | | VPG-1009 | Oligodendrocytes, rat | | VPI-1003 | Basic Neuron Nucleofector Kit | | | | | | Nucleofector Kits for Cell Lines | | VCA-1003 | Cell Line Kit V (for: C6, Neuro-2a, NG108-15, PC-12, SH-SY5Y, SK-N-SH) | | VCA-1005 | Cell Line Kit L (for: GH3, IMR-32) | | VCO-1001 | Cell Line Optimization Nucleofector Kit | | | | | | 96-well Nucleofector Kits for Primary Cells | | VHPI-1003 | Neurons, basic (96 reactions) | | VHPI-2003 | Neurons, basic (960 reactions) | | VHPG-1003 | Neurons, rat (96 reactions) | | VHPG-2003 | Neurons, rat (960 reactions) | | | | | | 96-well Nucleofector Kits for Cell Lines | | VHCA-1002 | Cell Line Kit SF (96 reactions) (for: Neuro-2a) | | VHCA-2002 | Cell Line Kit SF (960 reactions) (for: Neuro-2a) | | VHCO-1001 | Cell Line Optimization 96-well Nucleofector Kit |
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*Note: Due to the innovative pulse programs which are applied, SCN kits are compatible with Nucleofector II devices, Serial Version "S", only. Check here to make sure that you have this version of the device or simply contact our Scientific Support Team. For customers with an older version of the Nucleofector device who want to use these kits, amaxa offers a special device trade-in program at a reduced price.
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The Nucleofector Technology, comprising Nucleofection Process, Nucleofector Device, Nucleofector Solutions, Nucleofector 96-well Shuttle System and Nucleocuvette plates and modules is covered by patent and/or patent pending rights owned by amaxa AG.
amaxa, Nucleofector, nucleofection, maxGFP, 96-well Shuttle and Nucleocuvette are either registered trademarks or trademarks of amaxa AG in the U.S. and/or Germany and/or other countries.
amaxa disclaims all warranties, whether expressed or implied, including any warranty as to the quality, accuracy, safety, or suitability of the information provided in this e-newsletter for any particular purpose. Any use of the information contained on any page of this e-newsletter is evidence of agreement with these terms of use.
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