Figure 1.
Principle of oscillating nanomagnetic transfection.
Short interfering RNA (siRNA) or plasmid DNA is attached to magnetic nanoparticles and incubated with cells in culture (left). An oscillating magnet array below the surface of the cell culture plate pulls the particles into contact with the cell membrane (A) and drags the particles from side-to-side across the cells (B), mechanically stimulating endocytosis (C). Once the particle/nucleic acids complex is endocytosed, proton sponge effects rupture the endosome (D) releasing the nucleic acids (E) which either transcribes the target protein or silences the target genes (F) [3].
Figure 2.
Knockdown of GFP from GFP-HeLa cells.
Stably-expressing GFP-HeLa cells were transfected, either with a lipid reagent, nucleofection method or magnetofection using an oscillating magnet (2Hz frequency amplitude and 0.2 mm displacement), as indicated. Magnetofection using nTMag only was set as negative control (Mock). Fluorescent intensity (Median) was determined 72 hr post-transfection and knockdown levels were normalised to a scrambled control. Results are expressed as the mean±SD of at least three independent experiments. *, p<0.05; **, p≥0.05.
Figure 3.
Knockdown of Actin from HeLa and Rat Aortic Smooth Muscle Cells.
HeLa (A–C) or rat aortic smooth muscle cells (D, E) transfected with the either scrambled siRNA or siRNA against actin, as indicated, were seeded on six-well gelatine-coated plates 72 hrs post-transfection. Adherent, Hoechst–stained (nuclear) HeLa cells after transfection with scrambled siRNA (A) or actin siRNA (B) were counted by fluorescence microscopy, as described in the Methods section. Knockdown of actin in HeLa (C) or rat aortic smooth muscle cells (D) and its viability (E) was expressed as a percentage of the number of adherent cells transfected by actin siRNA over its scrambled control. Data represent the mean±SD of at least three independent experiments.
Figure 4.
Effects of inhibitors on transfection and cell viability.
HeLa cells were transfected with pEGFP-N1 in the presence of varying doses of inhibitors, as indicated and described in the Methods section. Transfection efficiency (A), viability (B) and Total Transfection (C) were determined 24 hr post-transfection using flow cytometry or MTT assay, respectively, and expressed as the mean±SD of at least three independent experiments.