Journal: Journal of Functional Biomaterials

Article Title: Using Magnetic Nanoparticles for Gene Transfer to Neural Stem Cells: Stem Cell Propagation Method Influences Outcomes

doi: 10.3390/jfb6020259

Figure Lengend Snippet: Effect of magnetofection with static ( F = 0 Hz) and oscillating ( F = 0.5–4 Hz) magnetic fields on transfection efficiency. ( A ) Representative phase image of monolayer cultures; ( B ) Representative double-merged image of DAPI-stained cultures at 48 h after Neuromag-mediated transfection with pmaxGFP conducted in the absence of a magnetic field; ( C ) Representative double-merged image of DAPI-stained cultures at 48 h after Neuromag-mediated transfection with pmaxGFP with an applied oscillating magnetic field of F = 4 Hz; ( D ) Bar chart showing proportions of transfected cells in NSC monolayers at 48 h after addition of Neuromag and pmaxGFP complexes with application of the indicated magnetic field.* P < 0.05 & *** P < 0.001 versus no magnetic field; +++ P < 0.001 versus static ( F = 0 Hz) magnetic field; n = 4 cultures (one-way ANOVA and Bonferroni’s MCT). Scale bar = 200 µm in (A, B & C).

Article Snippet: Neuromag MNPs were from Oz Biosciences (Marseilles, France), pmaxGFP plasmid (size 3.5 kb; encodes green fluorescent protein [GFP]) was from Amaxa Biosciences (Cologne, Germany) and pCMV-DsRed-Express2 plasmid (herein termed pDRE2; size 4.6 kb; encodes red fluorescent protein [RFP]) was from Clontech (Saint-Germain-en-Laye, France).

Techniques: Magnetofection, Transfection, Staining

Journal: Journal of Functional Biomaterials

Article Title: Using Magnetic Nanoparticles for Gene Transfer to Neural Stem Cells: Stem Cell Propagation Method Influences Outcomes

doi: 10.3390/jfb6020259

Figure Lengend Snippet: Long-term GFP expression in neurospheres derived from transfected monolayers. Monolayers were transfected with pmaxGFP with the indicated applied magnetic fields. At 48 h, cells were detached and passaged as neurospheres; spheres were dissociated and re-plated at weekly intervals. At the indicated times, the proportion of GFP expressing neurospheres and the extent of GFP expression (based on the proportion of cells within a sphere demonstrating GFP expression) were scored; categories for the latter were “low” (≤10% cells), “moderate” (11%–50% cells) and “high” (≥51% cells).

Article Snippet: Neuromag MNPs were from Oz Biosciences (Marseilles, France), pmaxGFP plasmid (size 3.5 kb; encodes green fluorescent protein [GFP]) was from Amaxa Biosciences (Cologne, Germany) and pCMV-DsRed-Express2 plasmid (herein termed pDRE2; size 4.6 kb; encodes red fluorescent protein [RFP]) was from Clontech (Saint-Germain-en-Laye, France).

Techniques: Expressing, Derivative Assay, Transfection, In Vitro

Journal: Journal of Functional Biomaterials

Article Title: Using Magnetic Nanoparticles for Gene Transfer to Neural Stem Cells: Stem Cell Propagation Method Influences Outcomes

doi: 10.3390/jfb6020259

Figure Lengend Snippet: Effects of transfection protocols on cell viability and neurosphere formation. Monolayers ( n = 4 cultures) were transfected with Neuromag-pmaxGFP complexes or with pmaxGFP only for controls, with application of the indicated magnetic fields. After 48 h, cells were detached from wells and a small proportion stained with trypan blue. ( A ) Bar chart showing the total number of cells per well. ( B ) Bar chart showing the proportion of viable cells. ( C ) Representative phase-contrast image of neurospheres formed from monolayers treated with particle/plasmid complexes; inset shows neurospheres formed from monolayers treated with plasmid only. ( D ) Fluorescence micrograph of neurospheres shown in (C), demonstrating GFP expression at 9 days post-transfection. ( E ) Bar chart showing the average sphere number per microscopic field. ( F ) Bar chart showing the average sphere diameter. Scale bar = 100 µm in (C,D).

Article Snippet: Neuromag MNPs were from Oz Biosciences (Marseilles, France), pmaxGFP plasmid (size 3.5 kb; encodes green fluorescent protein [GFP]) was from Amaxa Biosciences (Cologne, Germany) and pCMV-DsRed-Express2 plasmid (herein termed pDRE2; size 4.6 kb; encodes red fluorescent protein [RFP]) was from Clontech (Saint-Germain-en-Laye, France).

Techniques: Transfection, Staining, Plasmid Preparation, Fluorescence, Expressing

Journal: Journal of Functional Biomaterials

Article Title: Using Magnetic Nanoparticles for Gene Transfer to Neural Stem Cells: Stem Cell Propagation Method Influences Outcomes

doi: 10.3390/jfb6020259

Figure Lengend Snippet: MNP–mediated combinatorial gene delivery to NSC monolayers. Cultures ( n = 3) were magnetofected (oscillating magnetic field of F = 4 Hz) with complexes formed between Neuromag MNPs and either pDRE2, pmaxGFP or pDRE2 plus pmaxGFP (1:1 mix) plasmids; in all transfections, the final concentration of each plasmid was half that employed in the standard protocol. ( A ) Representative image of cells co-transfected with both plasmids. (A, insets) same field of cells in (A), showing GFP or RFP expression alone at 48 h post-transfection; ( B ) Bar chart showing the proportions of transfected cells that express GFP plus RFP, GFP alone and RFP alone after co-transfection of plasmids; ( C ) Bar chart showing transfection efficiencies for co-transfection and the corresponding single gene transfection controls. Scale bar = 20 µm in (A–C).

Article Snippet: Neuromag MNPs were from Oz Biosciences (Marseilles, France), pmaxGFP plasmid (size 3.5 kb; encodes green fluorescent protein [GFP]) was from Amaxa Biosciences (Cologne, Germany) and pCMV-DsRed-Express2 plasmid (herein termed pDRE2; size 4.6 kb; encodes red fluorescent protein [RFP]) was from Clontech (Saint-Germain-en-Laye, France).

Techniques: Transfection, Concentration Assay, Plasmid Preparation, Expressing, Cotransfection

Journal: Journal of Functional Biomaterials

Article Title: Using Magnetic Nanoparticles for Gene Transfer to Neural Stem Cells: Stem Cell Propagation Method Influences Outcomes

doi: 10.3390/jfb6020259

Figure Lengend Snippet: MNP-mediated delivery of a functional gene encoding FGF2—effect of magnetofection on transfection efficiency. Monolayers ( n = 3 cultures) were transfected with Neuromag complexed with either pFGF2-GFP, pAN-GFP (control plasmid lacking the FGF2 insert) or pmaxGFP (positive control), with application of the indicated magnetic fields, then studied at 48 h post-transfection. ( A ) Representative phase and fluorescence double-merged image of cells transfected with pFGF2-GFP, demonstrating nuclear expression of GFP. Inset is a representative image of cells transfected with pAN-GFP; note that GFP expression extends throughout the cytoplasm. ( B ) Bar chart showing the proportions of transfected NSCs under no magnetic field (none), static magnetic field (F0) and oscillating magnetic field ( F = 4 Hz; F4) conditions. * P < 0.05 and *** P < 0.001 for inter-field comparisons (indicated at top of chart) for a given plasmid; +++ P < 0.001 versus pmaxGFP for a given magnetic field condition (one-way ANOVA and Bonferroni’s MCT); n = 3 cultures. ( C ) Regression analysis demonstrating transfection efficiency is inversely related to plasmid size under no magnetic field (None; r 2 = 0.994; P < 0.05), static magnetic field (F0; r 2 = 0.998; P < 0.05) and oscillating ( F = 4 Hz) magnetic field (F4; r 2 = 0.999; P < 0.01) conditions. ( D ) Immunoblots sequentially probed with antibodies to FGF2 (top) and β-actin (loading control; bottom), demonstrating expression of a 60 kDa protein species (indicated by arrow) in extracts of cells ( n = 3 cultures) transfected with pFGF2-GFP (lanes 2, 4 and 6) but not with pAN-GFP (lanes 1, 3 and 5); the migration of size markers is displayed on the right-hand side. Scale bar = 5 µm in (A).

Article Snippet: Neuromag MNPs were from Oz Biosciences (Marseilles, France), pmaxGFP plasmid (size 3.5 kb; encodes green fluorescent protein [GFP]) was from Amaxa Biosciences (Cologne, Germany) and pCMV-DsRed-Express2 plasmid (herein termed pDRE2; size 4.6 kb; encodes red fluorescent protein [RFP]) was from Clontech (Saint-Germain-en-Laye, France).

Techniques: Functional Assay, Magnetofection, Transfection, Plasmid Preparation, Positive Control, Fluorescence, Expressing, Western Blot, Migration