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IBIAN Technologies le presenta: |
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MATra Examples and Applications |
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- Applications in Neurosciences
- Neurons
- Mouse fibrosarcoma
- FaDu carcinoma
- Hepatocellular carcinoma
- Primary hepatocytes
- VSa13
- Comparison of transfection rates of various cell lines
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MATra – best transfection practices for Neurosciences |
Neurosciences are a vast and expanding field of research focussing on highly sophisticated and enthralling questions. With Magnet Assisted Transfection IBA offers a very gentle and potent tool for the transfection of many kinds of neuronal cells. Magnet Assisted Transfection is the ideal solution to overcome problems related to the study of complex and easily interrupted systems.
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Transfection of APP into neuroblastoma using MATra
Cells maintain their endogenous expression pattern and stay unaffected from transfection related influences:
B103 neuroblastoma cells were plated at 105 cells/well in Dulbecco’s modified Eagle’s medium + 10% fetal calf serum on poly-L-ornithine-coated glass coverslips in 24-well plates (Corning Life Sciences, Lowell, MA) and transfected using 0.2–0.8 μg of plasmid DNA per well and MATra-A beads on a 24 Magnet Bar Plate. The medium was changed 1–2 hrs after transfection, and expression was allowed to proceed for a further 16–24 hrs.
Figure: Investigation of APP dimerization using APP-GFP. A, confocal image of a B103 cell expressing APP-GFP. B–G, wide-field images of B103 cells expressing APP-GFP alone (B–D) or in combination with APP-mCherry (E–G). B and E, GFP channel. C and F, mCherry channel. D and G, GFP lifetime. Scale bars: 10 μm. H, histograms of FRET efficiencies in different experimental conditions. PDF, probability density function.
Expression levels were high enough to aquire fluorescence lifetime images (Fig. B-G), which permitted calculating the levels of interaction between APP-GFP molecules in the cell (Fig. H)
Data kindly provided by Dr. Matthias Gralle, Max-Planck-Institute for Evolutionary Anthropology, Leipzig, Germany, Gralle et al. (2009) J Biol Chem 284, 15016-25. |
| "Several liposomal methods were tried out, but the transfection efficiency was low, and the transfected cells were rounded and visibly unhealthy. With MATra, the expression pattern of APP-GFP was indistinguishable from the known expression pattern of endogenous APP, and the cells maintained the typical elongated morphology with protrusions", said Dr. Gralle at MPI, Leipzig, Germany. |
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Neurons transfected with eGFP plasmid |
 Primary hippocampal neurons (E14) were grown on 15 mm glass coverslips on a 12 well at density of 150.000/cm². The neurons were transfected 4 d.i.v. with pSyn-eGFP using 25 µl MATra complex per well (prepared by adding a MATra-A Reagent-DNA complex mixture (2.8 µg cDNA; 2.8 µl beads) into 175 µl neuronal medium without serum). The cells were fixed 6 d.i.v. with 4% PFA and imaged.
"With MATra we can transfect and modulate the expression levels of exogenous proteins in highly sensitive primary neurons without any toxicity. Once optimized, double and even triple transfections with different DNA ratios are easily achieved" said Dr. Mika Ruonala, ENI, Göttingen.
(Data kindly provided by Dr. Mika Ruonala, Center for Membrane Proteomics, University of Frankfurt, Germany;
ruonala@em.uni-frankfurt.de)
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Mouse fibrosarcoma cells transfected with GFP plasmid |
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| Phase contrast |
GFP fluorescence |
Overlay |
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Magnet Assisted Transfection (MATra) of L929 fibrosarcoma cells. 1 x 105 L929 cells were seeded on poly-L-lysine coated glass coverslips and allowed to grow for 24 h. Subsequently, the cells were transfected with 1 µg of an expression vector coding for green fluorescent protein (GFP) as described in the standard protocol for MATra. 48 h after transfection, the cells were fixed with 4% (w/v) paraformaldehyde and expression of GFP was visualized by confocal laser scanning microscopy.
Transfection efficiency was 60 - 80% (see overlay).
(Data kindly provided by Dr. Lutz Thon and Dr. Dieter Adam, Institut für Immunologie, Universitätsklinikum Schleswig-Holstein Campus Kiel, Kiel, Germany)
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Transient transfection of stable carcinoma cells with GFP plasmid |
GFP expression in FaDu head and neck cancer cells after transient transfection with pGFP plasmid DNA. FaDu cells (5 x 105 cells per cavity of a 6 well plate) were transfected with 1.0 µg (B) or 1.5 µg (C) pGFP expression plasmid using MATra-A (1 µl/1 µg DNA). Control: 1.0 µg empty vector, transfected under same conditions (A). GFP fluorescence was detected by flow cytometry after 48 hours.
FaDu cells are typically transfected with standard lipofection reagents with an efficiency of about 10% (1 µg GFP in 5 x 105 cells in 6 wells).
With MATra expression of GFP was detected in 52.7% (1.0 µg) and 82.55% (1.5 µg) of the cells.
"With MATra we have been able to increase the transfection efficiency to rates as high as 80% at 48 h following treatment" stated Olivier Gires from the LMU Munich. "All cell lines tested showed an increased transfection rate with MATra-A in comparison to lipofection or electroporation protocols."
(Data kindly provided by Rauch, Schaffrik, Ahlemann and Gires, LMU Munich and GSF, Munich, Germany)
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With MATra transfection efficiency has been
increased 8x compared to lipofection. |
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Human hepatocellular carcinoma cells transfected with GFP plasmid
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| MATra-A |
Competitive lipofection reagent |
Magnet Assisted Transfection of hepatocellular carcinoma cells (Hep G2) was compared to lipofection. Transfections with pCG-IRES-GFP (own construct) were carried out in 96 well plates according to standard protocols without medium change. Cells were fixed with 2% PFA 24 hours post transfection for fluorescence microscopy. Confluency ~ 80 - 90%.
MATra shows much higher transfection efficiency than competitive lipofection reagent.
(Data kindly provided by Michael Schindler, University Ulm, Microbiology and Virology, Ulm, Germany)
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Magnet Assisted Transfection (MATra) of primary cultured rat hepatocytes |
"For primary cultured rat hepatocytes MATra has been the most efficient transfection method we have tried so far, and it is much more cost-effective than the common lipofection reagents on the market."
Prof. Dr. Okumura, Kansai Medical University, Osaka, Japan
 Hepatocytes prepared from liver were seeded on 3.5 cm diameter dishes (3-5x105 cells/dish) and allowed to grow overnight. The cells were transfected with pCMV-LacZ, a CMV enhancer/promoter-driven β-galactosidase plasmid, as described in the standard protocols for MATra. The cells were fixed with 1% glutaraldehyde and stained in 2 mg/ml X-Gal solution. β-galactosidase-expressing blue cells were examined by microscopy.
With MATra-A a minimum of 5% cells expressed β-galactosidase, which was several fold better than with lipofection.
(Data kindly provided by Prof. Mikio Nishizawa and Tadayoshi Okumura, Dept. Biomedica Sciences, Ritsumeikan University, Kusatsu, shiga, Japan).
See also transfection of hepatocytes with siRNA |
Magnet Assisted Lipofection of fish bone-derived VSa13 cells |
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Luciferase activity |
Conditions |
| A |
1 |
3 µl of Lipofection Reagent "F6"/500 ng GAL-LUC and 50 ng CMV-GAL |
| B |
7.59 |
like A, plus 1 µl of MA Lipofection Enhancer |
| C |
64.75 |
like B, but 2500 ng GAL-LUC and 250 ng CMV-GAL |
The fish bone-derived VSa13 cells were cultured in D-MEM supplemented with 10% FBS. Magnet Assisted Lipofection was performed in cultures at 60 - 80% confluence grown in 12 well plates and in absence of FBS. CMV-GAL and a GAL-LUC constructions were co-transfected using MA Lipofection Enhancer combined with Lipofection reagent "F6". (Alternatively, IBA's lipofection reagent IBAfect can be used!)
Especially for cells difficult to transfect, it is important to titrate the optimal DNA concentration to obtain highest transfection efficiencies. In VSal3 fish bone-derived cells, multiplying DNA quantity by 5 resulted in about 8.5-fold increase in transfection efficiency.
(Data kindly provided by Vincent Laizé, Universidade do Algarve, Faro, Portugal)
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Comparison of transfection rates of various cell lines |
| HeLa |
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| CHO-K1 |
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Standard transfection method |
Magnet Assisted Transfection |
For indicated cells the following methods were tested.
1: Calcium phosphate vs Magnet Assisted Lipofection,
2-4 Lipofection vs Magnet Assisted Lipofection, 5: Lipofection vs Magnet Assisted Transfection. Data kindly provided by industrial IBA customer. |
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Data kindly provided by Dr. Günther Keil,
Friedrich-Loeffler-Institute, Federal Research
Institute for Animal Health, Island Riems,
Germany. |
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