1. Poly-ornithine/Laminin (Po/L) Dish Coating
NOTE: Volumes are adapted to coat a 24-well plate.
<ol>
	<li>If needed, put a sterile 12 mm coverslip in the well.</li>
	<li>Coat the wells with 500 &#181;L of 10 &#181;g/mL Poly-L-Ornithine solution dissolved in water.</li>
	<li>Let the wells settle at room temperature for 1 h.</li>
	<li>Remove the Poly-L-Ornithine solution and air-dry for 30 min.</li>
	<li>Coat the wells overnight at 37 &#176;C with 500 &#181;L of 3 &#181;g/mL laminin in bicarbonate L-15. 
	NOTE: Wells can be stored at 37 &#176;C for 7 days in the incubator. For long incubations, adding sterile PBS between the wells can help to avoid medium evaporation.</li>
</ol>
2. Motor Neuron Culture
<ol>
	<li>Dilute the MNs to the appropriate density, usually 5,000 to 10,000 cells in 500 &#181;L of culture medium per well in a 24-well plate. 
	NOTE: Seeding density is around 30,000 and 1,500 cells for 6- and 96-well plates, respectively.</li>
	<li>Remove the laminin solution with a P1000.</li>
	<li>Immediately transfer the MNs diluted in culture medium into the coating plates to avoid drying.</li>
	<li>Incubate the culture for 2 days at 37 &#176;C.</li>
</ol>
3. Magnetofection of Motor Neurons
NOTE: In the following steps, the quantities used are meant for the transfection of one well of a 24-well plate. Please refer to the manufacturer's protocol for another format.
<ol>
	<li>For the DNA preparation, resuspend 1 &#181;g of DNA in 50 &#181;L of neuronal culture medium and vortex for 5 s. Here, 9 kb plasmids with a CAGGS promotor (pCAGEN) controlling neurofilament cDNA are used.</li>
	<li>For bead tube preparation, resuspend 1.5 &#181;L of beads in 50 &#181;L of neuronal culture medium.</li>
	<li>Add the 50 &#181;L bead solution to the 50 &#181;L DNA solution and incubate for 20 min at room temperature (total volume = 100 &#181;L).</li>
	<li>During this incubation, withdraw 100 &#181;L of culture medium from the well that will be transfected. 
	NOTE: Put the magnetic plate in the incubator to warm it to 37 &#176;C.</li>
	<li>Transfer 100 &#181;L of the DNA/bead mix to the well, and incubate the plate 20&#8211;30 min on the magnetic plate at 37 &#176;C.</li>
</ol>

<table><tbody><tr><td>Plasmid pCAGEN</td><td>Addgene</td><td>#11160</td><td></td></tr><tr><td>Neuron cell culture medium</td><td>ThermoFisher Scientific</td><td>A3582901</td><td>Neurobasal Plus medium</td></tr><tr><td>Poly-DL-Ornithine</td><td>Sigma-Aldrich</td><td>P8638</td><td></td></tr><tr><td>Laminin</td><td>Sigma-Aldrich</td><td>L2020</td><td></td></tr><tr><td>Sodium bicarbonate</td><td>ThermoFisher Scientific</td><td>25080094</td><td></td></tr><tr><td>L-15 medium</td><td>ThermoFisher Scientific</td><td>11415056</td><td></td></tr><tr><td>PBS w/o Ca Mg</td><td>ThermoFisher Scientific</td><td>14190144</td><td>Without Mg2+ Ca2+</td></tr><tr><td>Round coverslip</td><td>NeuVitro, Knittel glass</td><td>GG-12-Pre</td><td>12 mm</td></tr></tbody></table>

magnetofection-based transfection in vitro: a magnetic field-assisted technique to deliver plasmids into primary mouse neuronal cells using magnetic nanoparticles

Magnetofection-Based Transfection In Vitro: A Magnetic Field-Assisted Technique to Deliver Plasmids Into Primary Mouse Neuronal Cells Using Magnetic Nanoparticles

Source: Jacquier, A. et al. <a href='https://www.jove.com/v/57988'>Modeling Charcot-Marie-Tooth Disease In Vitro by Transfecting Mouse Primary Motoneurons.</a> J. Vis. Exp. (2019)
In this video, we demonstrate magnetically-guided transfection of plasmid DNA in primary neuronal cell culture. Magnetofection uses an external magnetic field to guide the delivery of plasmids bound to magnetic nanoparticles into cell cytoplasm.

Source: Jacquier, A. et al. Modeling Charcot-Marie-Tooth Disease In Vitro by Transfecting Mouse Primary Motoneurons. J. Vis. Exp. (2019)
In this video, we ...

Begin with fluorescent protein-encoding plasmid DNA or pDNA suspension in media. Add biocompatible, cationic polymer-coated magnetic nanoparticles to the pDNA suspension and incubate.
The positively-charged cationic polymers electrostatically interact and conjugate with negatively-charged pDNA, resulting in pDNA condensation and formation of 'magnetic polyplexes' with an overall positive charge.
Now, transfer the polyplex mixture into a multi-well plate containing adherent primary mouse neuronal cell culture. Mount the multi-well plate over a magnetic plate and incubate.
The magnetic field generated by the magnetic plate causes the polyplexes to sediment and accumulate over the neuronal cell surface. This close contact with the cell surface facilitates the non-specific binding of positively-charged polyplexes to negatively-charged cell membrane-associated proteoglycans.
Subsequently, the polyplexes get internalized via endocytosis - a process wherein the local regions of the cell membrane invaginate and pinch off to form membrane-bound vesicles called endosomes.
Inside the cytoplasm, the polyplex's cationic polymers induce proton accumulation in the endosome. The simultaneous influx of chloride ions to maintain charge neutrality increases endosomal ionic strength, leading to water influx.
Eventually, the endosomes swell and rupture due to osmotic pressure, releasing polyplexes into the cytoplasm. The condensed pDNA within the polyplexes facilitate increased cytosolic motility, while the cationic polymers protect pDNA from cytoplasmic nuclease degradation.
Following dissociation of the polyplexes, the liberated pDNA - during the appropriate cell-cycle phase when the nuclear membrane temporarily disassembles - get translocated to the nucleus. Cells successfully transfected with pDNA express fluorescent proteins.

To culture motor neurons, dilute them to 5,000 to 10,000 cells in 500 microliters of culture medium per well in a 24-well plate. Then, remove the laminate solution with a P1000 pipette tip. Immediately transfer the motor neurons diluted in culture medium to the coating plates to avoid drying.
To prepare DNA, resuspend 1 microgram of DNA in 50 microliters of neuronal culture medium, and vortex for 5 seconds. To prepare the bead tube, resuspend 1.5 microliters of beads in 50 microliters of neuronal culture medium. Add 50 microliters of bead solution to 50 microliters of DNA solution, and incubate for 20 minutes at room temperature.
During this incubation, withdraw 100 microliters of culture medium from the well that will be transfected. Subsequently, transfer 100 microliters of the DNA-bead mix to each well, and incubate the 24-well plate 20 to 30 minutes on the magnetic plate at 37 degrees Celsius.

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3.0K Views. Begin with fluorescent protein-encoding plasmid DNA or pDNA suspension in media. Add biocompatible, cationic polymer-coated magnetic nanoparticles to the pDNA suspension and incubate. The positively-charged cationic polymers electrostatically interact and conjugate with negatively-charged pDNA, resulting in pDNA condensation and formation of 'magnetic polyplexes' with an overall positive charge. Now, transfer the polyplex mixture into a multi-well plate containing adherent prima...

Magnetofection-Based Transfection In Vitro: A Magnetic Field-Assisted Technique to Deliver Plasmids Into Primary Mouse Neuronal Cells Using Magnetic Nanoparticles

In This Article

Abstract

Protocol

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Tags

Name	Company	Catalog Number	Comments
Plasmid pCAGEN	Addgene	#11160
Neuron cell culture medium	ThermoFisher Scientific	A3582901	Neurobasal Plus medium
Poly-DL-Ornithine	Sigma-Aldrich	P8638
Laminin	Sigma-Aldrich	L2020
Sodium bicarbonate	ThermoFisher Scientific	25080094
L-15 medium	ThermoFisher Scientific	11415056
PBS w/o Ca Mg	ThermoFisher Scientific	14190144	Without Mg2+ Ca2+
Round coverslip	NeuVitro, Knittel glass	GG-12-Pre	12 mm