NeuroMag

Protocol tips

Upstream tips	Protocol tips	Downstream tips
• DO NOT FREEZE THE MAGNETIC NANOPARTICLES! • DO NOT ADD ANYTHING TO THE STOCK SOLUTION OF NANOPARTICLES!

Upstream tips
• DO NOT FREEZE THE MAGNETIC NANOPARTICLES! • DO NOT ADD ANYTHING TO THE STOCK SOLUTION OF NANOPARTICLES!

Publication protocol

3.4. Magnetic Nanoparticle-Based DNA Transfection
Three plasmid reporter constructs containing green fluorescent protein (GFP) genes: pCMV-eGFP-SV40noDTS, pCMV-eGFP-SV40DTS or pCMV-eGFP-SMGA2294 were each suspended individually in 100 µL of Brainbits Transfection Medium. The plasmid DNA suspension solutions were then combined with γ-Fe2O3 nTMag (Nanotherics, Staffordshire, UK), PolyMag Neo (Oz Biosciences, San Diego, CA, USA) or Neuromag (Oz Biosciences, San Diego, CA, USA) magnetic nanoparticle vectors and then incubated for 15 min at r.t. (~23 °C). Ultimately, 7 of 9 possible nanoparticle-DNA conjugates were formed, since Polymag Neo-SMGA and nTMag-SMGA complexes were not produced for subsequent experimentation. As a positive control, Lipofectamine™-2000 (Life Technologies, Carlsbad, CA, USA) was also conjugated in parallel with the same reporter plasmids in a 20 min incubation at ca. 23 °C (r.t.). The mass of plasmid DNA for transfecting a single well in a 24-well cell culture plate was held constant at a value of 0.5 µg. The respective amount of vector conjugated with this amount of plasmid DNA was dependent on an empirically derived ratio (data not shown). Specifically, 1.25 µL Lipofectamine™-2000, 0.5 µL Polymag Neo, 0.5 µL nTMag and 1.7 µL Neuromag were conjugated with 0.5 µg of the noDTS, SV40DTS or SMGA plasmid. Next, complete media was used to increase conjugation reaction volumes to a working volume of 400 µL per culture plate well for transfection. Then, the wells of the 24-well culture media plate containing the SH-SY5Y cells for transfection were aspirated and replaced with 400 µL of the MNP-pDNA complex solution. Next, the culture plate was affixed to the Magnefect-nano II™ array sample holder and placed into a cell culture incubator for 30 min. Last, the oscillation program was initiated via the Magnefect-nano II™ controller unit, with the transfection parameters of 2 Hz frequency, 0.2 mm displacement and a duration of 3600 cycles or 30 min [4,7].

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Manufacturer protocol

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