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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Dorsolateral Telencephalon Microdissection
- Prepare the dissection medium (100 mL): 98.5 mL Hank's Balanced Salt Solution (HBSS), 0.5 mL 5 M 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) pH 7.4, 1 mL penicillin/streptomycin (10,000 units/mL and 10,000 µg/mL).
- Filter-sterilize the dissection medium.
- Remove embryonic day 14 (E14) embryos from a pregnant mouse C57/Bl6 (Mus musculus) under anesthesia with isoflurane.
NOTE: Consider E0 as the day of vaginal plug detection. Dissection should be completed no later than 1 h after removal from the pregnant mouse. This protocol also applies to embryos E11-E17.
- Remove the embryos by hysterectomy under sterile conditions.
- Transfer the embryos to a Petri dish with a cold dissection medium (4 °C).
- Remove the brains from the skull by cutting along the longitudinal fissure. The number of embryos usually varies from five to ten. Each E14 brain yields approximately 2 x 106 cells, including progenitors and postmitotic neurons (1:1 ratio)
NOTE: Use the stereomicroscope for the following steps.
- Remove the meninges using dissection forceps. Split the telencephalon in the middle to separate the hemispheres. To isolate the dorsolateral telencephalon, cut along the dorsomedial curve and pallial-subpallial boundary using dissection forceps. Transfer the dorsolateral telencephalon to a 2 mL tube with a cold dissection medium (4 °C) until all brains are dissected.
2. Cell Dissociation and Plating
- Prepare the proliferation medium (50 mL of DMEM+10% FCS): 44 mL Dulbecco Modified Eagle's Medium (DMEM), 5mL Fetal Calf Serum (FCS), 0.5 mL 40% glucose, 0.5 mL penicillin/streptomycin (10,000 units/mL and 10,000 µg/mL). Filter-sterilize the proliferation medium.
- Prepare differentiation medium (50 mL of DMEM + 2% B27): 48 mL DMEM, 1 mL B27, 0.5 mL 40% glucose, 0.5 mL penicillin/streptomycin (10,000 units/mL and 10,000 µg/mL). Filter-sterilize the differentiation medium.
- After micro-dissection of the dorsolateral telencephalon from E14 mice, centrifuge the tube with the collected tissue and cold dissection medium (for 5 min at 4 °C, 340 x g) to precipitate the tissue.
- Remove the supernatant with a pipette and add 1 mL of pre-warmed (37 °C) Trypsin-Ethylenediaminetetraacetic acid (EDTA) (0.05%) for chemical digestion. Incubate for 15 min at 37 °C. Add 2 mL of proliferation medium to stop trypsin activity.
- Polish the tip of a glass Pasteur pipette over a gas burner or Bunsen burner for a few seconds to slightly narrow the aperture. Wash the pipette by aspirating FCS to coat the tip. To avoid bubbles, dissociate the cells mechanically with a fire-polished and FCS-coated Pasteur pipette.
- Centrifuge the cells (for 5 min at 4 °C, 340 x g). Remove the supernatant with a pipette. Add 1 mL of proliferation medium and resuspend the cells using a pipette. Repeat this step once.
- Prepare a 1:1 dilution of the cell suspension using a 0.4% Trypan Blue solution. The non-viable cells will be blue. Using a Neubauer chamber, count the number of viable cells (unstained).
- Dilute the cells in a proliferation medium to get 106 cells/mL. Add 500 µL of the cell suspension to each well of a 24-well tissue culture plate (approximately 2.5 × 105 cells/cm2). Incubate the cells at 37 °C and 5% CO2.
NOTE: Do not use glass coverslips, as they can move during the experiment and change the field of observation. Alternatively, use glass bottom tissue culture plates.
NOTE: If you are not using cell-culture-treated multidishes, it is important to pre-treat your plates with Poly-D-Lysin (50 µg/ml) for 2 h at 37 °C. PDL-treated plates can be stored at 4 °C after washing with distilled water.