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Primary human endothelial cells transfected with GFP plasmid

Comparison of transfection rates. Primary human dermal microvascular endothelial cells (HDMEC) were transfected with a GFP reporter plasmid using Magnet-Assisted Transfection (MATra), our non-liposomal PromoFectin-HUVEC, and well-known, leading transfection reagents dedicated for primary cell transfection. The appropriate protocols recommended by the manufacturers were followed. All transfections were performed in triplicate.
Primary human umbilical vein endothelial cells (HUVEC) transfected with a GFP reporter plasmid.

Mouse fibrosarcoma cells transfected with GFP plasmid

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 hours 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 for IBA GmbH by Dr. Lutz Thon and Dr. Dieter Adam, Institut für Immunologie, Universitätsklinikum Schleswig-Holstein Campus Kiel, Kiel, Germany)

Phase contrast
GFP fluorescence
Overlay

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 (see graphic below).

"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 for IBA GmbH by Rauch, Schaffrik, Ahlemann and Gires, LMU Munich and GSF, Munich, Germany)

With MATra transfection efficiency has been increased 8x compared to lipofection.

  

Human hepatocellular carcinoma cells transfected with GFP plasmid

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 for IBA GmbH by Michael Schindler, University Ulm, Microbiology and Virology, Ulm, Germany)

 

MATra-A
Competitive lipofection reagent

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 for IBA GmbH by Dr. Mika Ruonala, European Neuroscience Institute, Göttingen, Germany; mika.ruonala@medizin.uni-goettingen.de)

 

 

Magnet Assisted Transfection (MATra) of primary cultured rat hepatocytes

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.

"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.

(Data kindly provided for IBA GmbH by Mikio Nishizawa and Tadayoshi Okumura, Dept. Medical Chemistry, Kansai Medical University, Osaka, Japan)

 

 

Magnet Assisted Lipofection of fish bone-derived VSa13 cells

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, any other lipofection reagent 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 for IBA GmbH by Vincent Laizé, Universidade do Algarve, Faro, Portugal)

  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

Insect Cells

"We have used MATra-A as an alternative to electroporation for Sf9 insect cell transfection with Baculovirus and have been excited about the performance: The MATra approach is extremely gentle and does not cause the cell death we often observe with the harsh electroporation procedures. Therefore, we now routinely use the MATra system for insect cell transfection."

Dr. Rudolf Hauptmann, Boehringer Ingelheim RCV GmbH & Co KG, Austria

 

Human endometrial stromal cells transfected with siRNA

Primary cultures of human endometrial stromal cells were plated in a 96 well plate at a density of 13,000 cells/well. Twenty-four hours later medium was changed. Fluorescein-siRNA was diluted in OptiMEM® I Reduced Serum Medium (GIBCO) to 0.9 µg/108 µl. MATra-A reagent (0.45 µl) was added to obtain a ratio of 2:1 (siRNA:MATra-A). After 20 minutes incubation at room temperature, 15 µl of the mixture (corresponding to 125 ng siRNA) were added per well. The culture plate was placed on a 96 Magnet Plate for 15 minutes at 37°C. Cells were incubated for 20 hours at 37°C, medium was changed before microphotographs were taken. Virtually all cells had taken up the fluorescent siRNA.
(Data kindly provided for IBA GmbH by Dr. Birgit Gellersen, Endokrinologikum Hamburg, Hamburg, Germany)
 

Transfection of Carcinoma Cell Lines with siRNA

Efficient transient transfection of siRNA in head and neck cancer cells. The cell line ANT-1 was transiently transfected with MATra-A (1 µl/µg DNA) in a 6 well format (5 x 105 cells/cavity) with siRNA against protein 1 (100 nM). After 24 hours total RNA was isolated and expression of protein 1-specific mRNA determined by RT-PCR (upper lane). siRNA 13 are three different oligonucleotide sequences. Control for consistent loading and cDNA quality: expression of ubiquitary GAPDH mRNA (lower lane). Protein 2 expression in head and neck cancer cells GHD-1. GHD-1 cells (5 x 105 cells/cavity of a 6 well plate) were transiently transfected with two different siRNAs against protein 2. Expression of protein 2 was detected with specific antibodies in an immunoblot 72 hours after transfection with MATra-A (1 µl /µg DNA). As control ubiquitary β-actin was detected as well. Treating the carcinoma cells with specific siRNA caused a clear inhibition of protein 1/protein 2 expression which indicates high transfection efficiencies.


(Data kindly provided for IBA GmbH by Rauch, Schaffrik, Ahlemann and Gires, LMU Munich and GSF, Munich, Germany)
"After having tested MATra in a variety of experimental set ups we can summarize the following advantages:

  • high transfection efficiency
  • easier to handle
  • high reproducibility
  • serum compatibility
  • low sensibility against cell confluence"

Dr. Oliver Gires, LMU Munich, Germany.


Greatly improved transfection rates with commonly used cell lines!

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 customer.
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