Biosafety classification is based on U.S. Public Health Service Guidelines, it is the responsibility of the customer to ensure that their facilities comply with biosafety regulations for their own country.

The neuroepithelial cell lines, NE-4C (TS209401) and NE-GFP-4C (ATCC CRL-2926) were established from the cerebral vesicles of 9-day-old mouse embryos lacking the functional p53 genes. xad

Sox-2, Otx-2, En-1

Volumes used in this protocol are for 75 cm² flasks; proportionally reduce or increase amount of dissociation medium for culture vessels of other sizes.

1. Remove and discard culture medium.
2. Briefly rinse the cell layer with Ca++/Mg++ free Dulbeccos phosphate-buffered saline (D-PBS) or 0.05% (w/v) Trypsin - 0.53 mM EDTA solution to remove all traces of serum which contains trypsin inhibitor.
3. Add 2.0 to 3.0 mL of Trypsin-EDTA solution to flask and observe cells under an inverted microscope until cell layer is dispersed (usually within 5 to 15 minutes).
   Note: To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. Cells that are difficult to detach may be placed atxa037°C to facilitate dispersal.
4. Add 6.0 to 8.0 mL of complete growth medium and aspirate cells by gently pipetting.
5. Transfer cell suspension to a centrifuge tube and spin at approximately 125 X g for 5 to 10 minutes. Discard supernatant.
6. Resuspend the cell pellet in fresh growth medium. Add appropriate aliquots of the cell suspension to new poly-L-lysine coated culture vessels. An inoculum of 2 X 10⁴ to 4 X 10⁴ viable cells/cm² is recommended.
7. Incubate cultures at 37°C. Subculture when cell concentration is between 3 X 10⁵ and 4 X 10⁵ cells/cm².

Medium renewal: Every 2 to 3 days.

Freeze medium: Culture medium, 90%; DMSO,10%

Storage temperature: liquid nitrogen vapor phase

Atmosphere: 5% CO₂ in air recommended

Temperature: 37°C