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1. Poly-ornithine/Laminin (Po/L) Dish Coating
NOTE: Volumes are adapted to coat a 24-well plate.
- If needed, put a sterile 12 mm coverslip in the well.
- Coat the wells with 500 µL of 10 µg/mL Poly-L-Ornithine solution dissolved in water.
- Let the wells settle at room temperature for 1 h.
- Remove the Poly-L-Ornithine solution and air-dry for 30 min.
- 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
- 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.
- Remove the laminin solution with a P1000.
- Immediately transfer the MNs diluted in culture medium into the coating plates to avoid drying.
- 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.
- 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.
- For bead tube preparation, resuspend 1.5 µL of beads in 50 µL of neuronal culture medium.
- Add the 50 µL bead solution to the 50 µL DNA solution and incubate for 20 min at room temperature (total volume = 100 µL).
- 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.
- 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.