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1. Neural stem cell induction
NOTE: Depending on cell conditions, about 5 or 7 days after transfection, monolayer adherent cells will reach 30 - 50% confluence (Figure 1C).
- Remove the supernatants containing floating cells and spheres and then add 1 ml of neural progenitor medium (Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F12), containing 1x N2 supplement, 0.1% (w/v) bovine serum albumin (BSA), 1% (v/v) antibiotics, 20 ng/ml of basic fibroblast growth factor (bFGF), and 20 ng/ml of epidermal growth factor (EGF)) into the adherent cells.
- Optionally, dissociate the cell spheres by gently pipetting and centrifuging the cells at 170 x g for 10 min. Remove the supernatant and resuspend the cells in 1 ml of neural progenitor medium in a new 24-well plate coated with matrigel per instruction as a backup.
- Change the medium every other day till the cells reach 60 - 80% confluent, usually after 1 week.
- Discard the supernatant and add in 1 ml of neural stem cell medium (serum-free medium, NSC SFM). Dissociate the cells using a cell scraper, followed by very gentle pipetting.
- Remove the cells and seed all of them into one well of a 6-well plate. Add another 1 ml of neural stem cell medium to make 2 ml media for each well. Incubate the cells at 37 °C in a 5% CO2 incubator.
- Change the medium every other day.
- When cells reach 60% confluence, dissociate and replate the cells at a 1:3 ratio following the steps in 1.4 and 1.6.
