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Adherent cells - plasmids

Primary rat hepatocytes

Matra has been efficiently used for 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 by Prof. Mikio Nishizawa and Tadayoshi Okumura, Dept. Biomedica Sciences, Ritsumeikan University, Kusatsu, Shiga, Japan).

Primary cortical neurons

Embryonic cortical neurons were transfected with human NCAM. After transfection membrane-localized NCAM (not endocytosed) was detected using a Cy3-coupled secondary antibody (red). Afterwards, the internalised, endocytosed NCAM was stained by a Cy2-coupled secondary antibody (green, see arrows) in the cell soma (left) and in axonal vesicles (right).

Details see application note (pdf, 55 kb) and reference article “Renker et al. MATra- ein Trojanisches Pferd für eine zellschonende Transfektion, BIOSpektrum 2010“ (pdf, 1819 kb)

Primary cortical neurons from mice embryonic day 15.5 (E15.5) were grown on poly-L-lysine coated coverslips at a density of 800.000 cells/well in a 24-well plate. The neurons were transfected after 1 day in vitro (DIV 1) with pCX-EGFP-N1 plasmid. Transfection was carried out as recommended by the manufacturer
(0.6 µg DNA, 0.6 µL Matra-A reagent). Cells were fixed 24 h later (DIV 2) and GFP fluorescence was visualized using a confocal laser scanning microscope.

"With MATra we achieved a higher transfection efficiency than with different liposomal transfection methods and no toxicity to the cells was observed."
Dr. Simone Diestel, Institute of Animal Science, University Bonn, Germany

Primary hippocampal neurons (E14)

Primary hippocampal neurons transfection with MATra

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, Center for Membrane Proteomics, University of Frankfurt.

(Data kindly provided by Dr. Mika Ruonala, Center for Membrane Proteomics, University of Frankfurt, Germany; ruonala@em.uni-frankfurt.de)

Cerebellar granular cells

Cotransfection of cultured cerebellar granular cells (CGCs) with a mix of four different vectors in a one-tube reaction using MATra.     

CGCs from CD1 mice were grown on glass coverslips in a 12 well plate over night. For transfection, a premixture of the four different expression vectors MyrPalm-mCFP, Actin-RFP, Flotillin-2-eYFP and Battenin-Alexa647 (400 ng in total, 100 ng each) was mixed with the MATra-A reagent in one tube and added to the cells. Without medium change, live cells were imaged with confocal scanning laser microscopy running in sensitized emission FRET mode 24 h post transfection. The successful transfection of Battenin-Alexa647 was detected post mortem.

Cultured cerebellar granular cells from CD1 mice were transfected by below 4 constructs (A-D) using MATra-A.

(A) MyrPalm-mCFP, cyan (provided by Dr. R. Tsien, UCLA)
(B) Actin-DsRed, red
(C) Flotillin-2-mVenus, yellow (B and C provided by Dr. R. Tikkanen, University of Giessen)
(D) Battenin-myc, detected by using GAM-Alexa647, dark green
(E) Surface: Crop of the whole image with 3D surface rendered fluorescence signals overlayed on phase contrast image.
(F) PhaCo: Phase contrast image

(Data kindly provided by Dr. Mika Ruonala, Center for Membrane Proteomics, University of Frankfurt, Germany.)

See application note (pdf, 1100 kb) and reference article “Renker et al. MATra- ein Trojanisches Pferd für eine zellschonende Transfektion, BIOSpektrum 2010“(pdf, 1819 kb)

Cervical carcinoma cells (HeLa)

Homologous recombination reporter-assay in HeLa cells using Magnet Assisted Transfection (MATra)

To analyze potential proteins involved in homologous recombination in HeLa pGC cells the corresponding endogenous protein was knocked down and an expression vector was created with the gene sequence of knocked down protein fused to the red fluorescent protein (RFP) gene sequence. HeLa pGC cells harboring a stably integrated homologous recombination substrate (GFP gene sequence with an I-SceI restriction site) were seeded on glass coverslips and allowed to grow for 24 h. Subsequently, the cells were cotransfected with the expression vector coding for the RFP fusion protein and an expression vector coding for the double-strand break inducing endonuclease I-SceI. For this purpose both expression vectors and MATra-A were mixed in one single tube and after incubation these complexes were added to the cells. Without medium change, the cells were fixed with 2.5% FA/PBS and stained with an anti-GFP and an anti-RFP antibody 72 h after transfection. Expression of RFP and GFP were visualized by microscopy. Reconstitution of the I-SceI induced double-strand break of the GFP sequence by homologous recombination can be verified by GFP-positive cells.

(A) Red fluorescent protein (RFP) indicating the expression of the knocked down protein in HeLa pGC cells.
(B) GFP-positive cells indicating homologous recombination.
(C) Merge showing double transfectants in yellow.

"With MATra we have been able to strongly increase our transfection efficiency."
Olivia Barton, Technical University, Darmstadt, Germany

(Data kindly provided by Olivia Barton, Technical University, Darmstadt, Germany)

MATra in comparison to lipofection

Magnet Assisted Transfection (MATra) of HeLa cells was compared to lipofection. HeLa cells were seeded on glass coverslips and allowed to grow for 24 h. Subsequently, the cells were transfected with a 7.5 kb expression vector coding for green fluorescent protein (GFP) as described in the standard protocol for MATra. Without medium change, the cells were fixed with 2.5% FA/PBS 48 h after transfection, stained with an anti-GFP antibody, and expression of GFP was visualized by microscopy.

(A) MATra-A
(B) Competitive reagent

HeLa cells showed an increased transfection rate with MATra in comparison to lipofection. Cytotoxicity could not be observed although no medium change was performed after transfection.

(Data kindly provided by Olivia Barton, Technical University, Darmstadt, Germany)

Magnet Assisted Transfection (MATra) of HeLa cells with GFP-fusion proteins

HeLas were seeded in cell culture dishes and allowed to grow for 24 h. Subsequently, cells were transfected with different GFP-fusion protein expression vectors. Without medium change, cells were harvested and lysed with RIPA buffer 48 h after transfection. The GFP-fusion proteins were immunoprecipitated and in vitro kinase assays were performed.

Western Blot analysis of one GFP-fusion protein

"With MATra we have been able to increase the transfection efficiency and therefore could perform in vitro kinase assays using less cell extract than with previous transfection methods"
Olivia Barton, Technical University, Darmstadt, Germany

(Data kindly provided by Olivia Barton, Technical University, Darmstadt, Germany)

Neuroblastoma cells (B103)

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).

"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, now at Univ. Federal do Rio de Janeiro, Brazil.

(Data kindly provided by Dr. Matthias Gralle, now Univ. Federal do Rio de Janeiro, Brazil; Gralle et al. (2009) J Biol Chem 284, 15016-25.)

Choriocarcinoma cells (BeWo)

BeWo cells were efficiently transfected with an expression vector coding for green fluorescent protein (GFP) using magnet assisted transfection.
Therefore, BeWo cells were seeded 24 h before transfection in a 12well plate at a density of 180 000 cells/well. 1µg, 2µg and 3µg of plasmid respectively were incubated in 200µl OptiMEM® I Reduced Serum Medium with Lipofectamine in a ratio of 1:3 for15 minutes. MA Lipofection Enhancer reagent was added in a ratio 1: 1.2 with an additional incubation time of 15 minutes. The plate was placed on the MATra Magnet Plate for 15 minutes at room temperature to allow beads to penetrate the target cells. GFP fluorescence was detected by flow cytometry after 36 hours.
(Data kindly provided by Andrea Milenkovic, Human Genetics Regensburg, Germany)

Fig.: FACs analysis of GFP transfected BeWo cells

  1.  not transfected
  2.  1µg Plasmid + 3µl Lipofectamine + 1.2µl MA Lipofection Enhancer
  3.  2µg Plasmid + 6µl Lipofectamine + 2.4µl MA Lipofection Enhancer
  4.  3µg Plasmid + 9µl Lipofectamine + 3.6µl MA Lipofection Enhancer
  Tranfection efficiency %
Lipofectamine < 1
Lipofectamine+Enhancer 1µg Plasmid 13.8
Lipofectamine+Enhancer 2µg Plasmid 21.2
Lipofectamine+Enhancer 3µg Plasmid 35.3

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)

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 the competitive lipofection reagent.

(Data kindly provided by Michael Schindler, University Ulm, Microbiology and Virology, Ulm, Germany)

Head and neck cancer cells (FaDu)

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."

Transfection efficiency in FaDu cells is 8x increased when transfection is performed with matra.

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

(Data kindly provided by Rauch, Schaffrik, Ahlemann and Gires, LMU Munich and GSF, Munich, Germany)

Fish bone-derived cells (VSa13)

Matra is a hihgly efficient transfection tool especially for cells difficult to transfect.

  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)

Adherent cells - siRNA & oligos

Primary rat hepatocytes

Knockdown of inducible nitric oxide synthase (iNOS) mRNA by sense ODN and siRNA: Using MATra-A Reagents, our group has successfully introduced plasmid DNA and oligodeoxyribonucleotides (ODNs) into primary cultured rat hepatocytes and murine RAW264 macrophage cells [1–7].  Sense ODNs (i.e., single-stranded DNA) to iNOS mRNA inhibit the interaction between the iNOS mRNA and antisense transcript to knockdown iNOS mRNA [8].  To compare degradation efficacy of the sense ODN and siRNA (i.e., double-stranded RNA), we transfected rat hepatocytes and estimated iNOS mRNA levels by reverse transcription and real-time PCR.  As shown in Fig. 1, the sense ODN S4 corresponding to iNOS gene exon 27 [8] reduced the level of iNOS mRNA to 63.9% to that of mock transfection.  We designed siRNAs (siE26 and siE27) corresponding to iNOS gene exon 26 and 27, respectively.  When they were introduced, iNOS mRNA was reduced to the levels which were comparable to that when S4 was introduced.  Considering that the transfection efficiency is about 50 %, it demonstrated that both siRNAs and the sense single-stranded ODN to iNOS mRNA are successfully introduced to rat hepatocytes and effectively degrade iNOS mRNA.

"MATra is a powerful tool to transfect difficult cells, such as primary cultured rat hepatocytes, with ALL the types of DNA/RNA including plasmid DNA, ODN, and siRNA."

Prof. Dr. Nishizawa, Ritsumeikan University, Japan

References
1) Tanaka H, et al. J Hepatol. 48:289–299 (2008). 
2) Yoshida H, et al. Nitric Oxide. 18:105–112 (2008). 
3) Tokuhara K, et al. Nitric Oxide. 18:28–36 (2008). 
4) Habara K, et al. Nitric Oxide. 18:19–27 (2008). 
5) Matsui K, et al. JPEN J Parenter Enteral Nutr. 31:373–380 (2007). 
6) Ozaki T, et al. JPEN J Parenter Enteral Nutr. 31:366–371 (2007). 
7) Yoshida H, et al. Shock. 30:734–739 (2008).  
8) Matsui K, et al. Hepatology. 47:686–697 (2008).

Matra is a powerful tool to transfect DNA, RNA, ODN and siRNA into primary cultured rat hepatocytes.

Optimisation of c-jun over-expression in primary human chondrocytes Method. Primary human articular chondrocytes isolated from cartilage of patients undergoing total knee replacement were cultured for approximately 10 d before trypsinisation and re-seeding into 6-well dishes at approx. 70% confluence. Cells were transfected with pCMV2 harbouring human c-jun or empty vector using either FuGENE HD or MATra-A transfection reagents, using protocols and a range of DNA:reagent ratios as suggested by manufacturers. After 24 h cells were lysed and 10 μg lysates subjected to SDS-PAGE and immunoblotting using α-Jun antibody (Santa Cruz).

“Our study of the molecular processes underlying cartilage degradation in arthritis has sometimes been hampered by poor transfection efficiency of primary human chondrocytes, despite our trialling a variety of liposomal transfection reagents. The increased efficiencies we have observed using MATra promises to expand the range of experiments we can attempt, significantly facilitating our research.”

Gary Litherland, Musculoskeletal Research Group, Institute of Cellular Medicine, Newcastle University, Newcastle, UK

Both a sense ODN and siRNAs effectively degrade iNOS mRNA. Rat hepatocytes (1.2 x 106 cells/well) were transfected with 1 mg ODNs (S4 and Scr4) or siRNAs (siE26, siE27, and siScr) using 1 mL MATra-A Reagent. Next day, total RNA was extracted from IL-1b-treated hepatocytes and subjected to real-time PCR of iNOS and EF-1a mRNA (n=3), as previously described [8]. Expression levels of iNOS mRNA were normalized by those of EF-1a mRNA. (–), mock transfection. S4, 5’-GTGTATAATTCCTTGATGAA-3’; Scr4, a scrambled control of S4, 5’-GATTGTTACTTAGAGACTAT-3’; siE26, 5’-CCAGCUCAAGAGCCAGAAAdTdT-3’ (sense strand is shown); siE27, 5’-CCGUGUAUAAUUCCUUGAUdTdT-3’; and siScr, a scramble control of siE27, 5’-GCUUGCUAUGUACACUUAUdTdT-3’.

(Data kindly provided by Mikio NISHIZAWA, Prof., Dr.
College of Life Sciences, Ritsumeikan University.  Kusatsu, Shiga, Japan.  e-Mail: nishizaw@sk.ritsumei.ac.jp)

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 by Dr. Birgit Gellersen, Endokrinologikum Hamburg, Hamburg, Germany)

Head and neck cancer cells (ANT-1)

Efficient transient transfection of siRNA in head and neck cancer cells. The cell line ANT-1 was transiently transfected with MATra-A (1 µl/1 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 / 1 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 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

Human umbilical vein endothelial cells (HUVEC)

80% down-regulation of the protein! Human umbilical vein endothelial cells (HUVEC) were efficiently transfected with short interfering RNA (siRNA) using magnet assisted transfection. Briefly, 3 µg of siRNA were diluted in OPTIMEM I medium to give a final volume of 200 μl for each well to be transfected (final concentration approximately 30 nM). For the formation of the transfection complex, 3 μl of MATra-si reagent were added to the diluted siRNA, carefully mixed and allowed to incubate at room temperature for 25 minutes. HUVEC were once washed in OPTIMEM I, the medium was discarded and then fresh OPTIMEM I (2 ml per well) was added to the cells. The siRNA/magnetic beads were then overlaid drop wise onto the cells (200 μl/well). Cells were incubated with the transfection complex on a custom made magnetic plate  which was specifically designed to fit the wells of Flexcell plates (Universal Magnet Plate, IBA) for 15 minutes in the cell incubator to allow beads to penetrate the target cells. Thereafter, to induce rapid zyxin turn over, cells were shortly exposed to cyclic stretch (30 minutes, 0.5 Hz, 10 % elongation1). After this manoeuvre, the medium was changed to prevent cytotoxic effects from the transfection reagent, and the cells were kept further under their normal culture conditions. Gene silencing was optimal 72 hours after transfection.

Other gene products knocked down with similar efficiencies: Ku70/Ku80, PARP1, hnRNP U.

Figure above: Zyxin knock down by Magnet Assisted Transfection (MATra). Immunofluorescence analysis of HUVEC. Representative confocal immunofluorescence images of zyxin (Cy3/red) in non-transfected human cultured endothelial cells (HUVEC; left picture) and HUVEC after Magnet Assisted Transfection (as described in the text; right picture). The cells were counter-stained with the focal adhesion protein paxillin (Cy2/green) and the DNA-binding nuclear dye DAPI (blue). Paxillin co-localizes with zyxin exclusively in the focal adhesions (yellow) but not in the stress fibers or in the nucleus.

 

Comparison of siRNA transfection into HUVEC using MATra.

 

"We have tried to efficiently transfect siRNA into Huvec for month with only minor success (up to 8% efficiency). With Magnet Assisted Transfection using the MATra reagent we finally achieved a knock down of 80% – definitely a break through."

PD Dr. Marco Cattaruzza, Institute for Physiology and Pathophysiology, Heidelberg University, Germany

1Zyxin is a very stable protein, thus, an efficient knock down is difficult to achieve. Under conditions of mechanical strain, the protein translocates to the nucleus where it regulates gene expression. In the nucleus, however, zyxin is by far less stable, i.e., it seems to undergo accelerated export and degradation. Therefore, stretching the cells is a zyxin-specific manoeuvre aiming at rapidly decreasing the pre-transfection levels of zyxin. In our hands, when attempting to knock down stable proteins, it is a good idea to try to accelerate their turn over, e.g., by (shortly) activating cellular pathways involving the protein in question.


Suspension cells

B-lymphoblastoid cells (Namalwa)

Effective knockdown of IFN-α1 AS RNA by sense oligodeoxynucleotides (seODNs) using MATra.

Non-adherent Namalwa cells were mixed with MATra-S Immobilizer to make them adherent. seODNs (S1, S2, S3, S4, Nc1, Nc2) coding for different regions of the IFN-α1 mRNA were incubated with MATra-A and added to the immobilized B-lymphoblastoid cells in order to knockdown IFN-a1 AS RNA expression. Next day, total RNA was extracted and the constitutive expression levels of IFN-α1 AS RNA were determined by RT-PCR. Only S1 seODN managed to knockdown the constitutive expression of IFN-a1 AS RNA.

Fig. Silencing of IFN-α1 AS RNA by sense ODNs.
From `Stabilization of human interferon- α1 mRNA by its antisense RNA´. Tominori Kimura, Shiwen Jiang, Mikio Nishizawa, Emi Yoshigai, Iwao Hashimoto, Masao Nishikawa, Tadayoshi Okumura, and Hisao Yamada. Cell. Mol. Life Sci. 70 (8): 1451-1467, 2013. DOI 10.1007/s00018-012-1216-x

"The combination of MATra-S and MATra-A system have worked so far best in our hands to introduce oligodeoxynucleotides and oligoribonucleotides into non-adherent cells, such as Namalwa cells."
Tominori Kimura, Professor for Microbiology and Cell Biology, College of Pharmaceutical Sciences, Ritsumeikan University, Japan

Reference:
Kimura et al., 2013. Cell. Mol. Life Sci. 70 (8): 1451-1467, 2013. DOI 10.1007/s00018-012-1216-x

Acute myeloid leukemia cells (THP-1)

Transient transfection of the monocytic cell line THP1 cells: GFP expression in THP1 cells after transient transfection with pCMV-ASC-GFP DNA (Origene). THP1 cells (1, 0.5 or 0.25 x 106 cells per well in a 12 well plate) were immobilized on collagen coated plates (BD) for 24h. The cells were transfected with 1.5 µg DNA expression plasmid using MATra-A (1 µl/1 µg DNA) or Lipofectamine and MA Lipofection enhancer. GFP fluorescence was detected by flow cytometry after 22 hours.

Transfection efficiency was increased in cells transfected with MatraA compared to cells transfected with Lipofectamine and had an optimal cell density between 0.5x106 and 0.25x106 (table 1). Figure 1 demonstrates a representative histogram of cells seeded with a density of 0.5x106 and transfected with Matra-A. 

Cell density Lipofectamine MatraA
1x106 2.5 20.2
0.5x106 4.5 26.3
0.25x106 4.4 17.4
Transfection of acute myeloid leukemia cells-THP-1 using MATra

Figure 1. Cell sample with density of 0.5x106 and transfected with MatraA (dotted line) compared to untransfected cells (full line). Transfection efficiency 28%

(Date kindly provided by Dahl, TB and Ranheim T Institute of Internal Medicine, Oslo University Hospital Rikshospitalet.)

Insect cells (SF9)

"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