• additional 2mM L-Glutamine
  • fetal bovine serum (FBS) to a final concentration of 10%
  
  

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/cm^2xa0 is recommended.
7. Incubate cultures at 37°C. Subculture when cell concentration is between 3 X 10⁵ and 4 X 10⁵ cells/cm² .

Temperature: 37°C

Atmosphere:xa0air, 95%; carbon dioxide (CO₂), 5%

Schlett K. et al. Retinoic acid induced neural differentiation in a neuroectodermal cell line immortalized by p53 deficiency. J. Neurosci. Res. 15;47(4):405-415 (1997) PubMed: 9057134

Demeter, K et al. Fate of cloned embryonic neuroectodermal cells implanted into the adult, newborn and embryonic forebrain. Exp. Neurol.188(2):254-267 (2004) PubMed: 15246825