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

프로토콜

1. Poly-ornithine/Laminin (Po/L) Dish Coating

NOTE: Volumes are adapted to coat a 24-well plate.

1. If needed, put a sterile 12 mm coverslip in the well.
2. Coat the wells with 500 µL of 10 µg/mL Poly-L-Ornithine solution dissolved in water.
3. Let the wells settle at room temperature for 1 h.
4. Remove the Poly-L-Ornithine solution and air-dry for 30 min.
5. Coat the wells overnight at 37 °C with 500 µL of 3 µg/mL laminin in bicarbonate L-15.
   NOTE: Wells can be stored at 37 °C for 7 days in the incubator. For long incubations, adding sterile PBS between the wells can help to avoid medium evaporation.

2. Motor Neuron Culture

1. Dilute the MNs to the appropriate density, usually 5,000 to 10,000 cells in 500 µL 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.
2. Remove the laminin solution with a P1000.
3. Immediately transfer the MNs diluted in culture medium into the coating plates to avoid drying.
4. Incubate the culture for 2 days at 37 °C.

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.

1. For the DNA preparation, resuspend 1 µg of DNA in 50 µL of neuronal culture medium and vortex for 5 s. Here, 9 kb plasmids with a CAGGS promotor (pCAGEN) controlling neurofilament cDNA are used.
2. For bead tube preparation, resuspend 1.5 µL of beads in 50 µL of neuronal culture medium.
3. Add the 50 µL bead solution to the 50 µL DNA solution and incubate for 20 min at room temperature (total volume = 100 µL).
4. During this incubation, withdraw 100 µL of culture medium from the well that will be transfected.
   NOTE: Put the magnetic plate in the incubator to warm it to 37 °C.
5. Transfer 100 µL of the DNA/bead mix to the well, and incubate the plate 20–30 min on the magnetic plate at 37 °C.

자료

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