1. Remove and discard culture medium.
2. Briefly rinse the cell layer with 0.25% (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 at 37°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 culture vessels. An inoculum of 5 X 10³ to 7 X 10³ viable cells/cm² is recommended.
7. Incubate cultures at 37°C. Subculture when cell concentration is between 1 X 10⁴ and 2 X 10⁴ cells/cm²

1. Upon receipt, visually examine the culture for macroscopic evidence of any microbial contamination. Using an inverted microscope (preferably equipped with phase-contrast optics), carefully check for any evidence of microbial contamination.xa0 Also, check to determine if the majority of cells are still attached to the bottom of the flask; during shipping the cultures are sometimes handled roughly and many of the cells often detach and become suspended in the culture medium (but are still viable).
2. If the cells are still attached, aseptically remove all but 5 to 10 mL of the shipping medium. The shipping medium can be saved for reuse. Incubate the cells at 37°C in a 5% CO₂ in air atmosphere until they are ready to be subcultured.
3. If the cells are not attached, aseptically remove the entire contents of the flask and centrifuge at 125 x g for 5 to 10 minutes. Remove shipping medium and save.xa0 Resuspend the pelleted cells in 10 mL of this medium and add to 25 cm² flask.xa0 Incubate at 37°C in a 5% CO₂ in air atmosphere until cells are ready to be subcultured.

Fieber LA, et al. Delayed rectifier K currents in NF1 Schwann cells. Pharmacological block inhibits proliferation. Neurobiol. Dis. 13: 136-146, 2003. PubMed: 12828937

Li Y, et al. Notch and Schwann cell transformation. Oncogene 23: 1146-1152, 2004. PubMed: 14762442