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Never allow culture to become completely confluent. Subculture when 80% confluent or less.

Volumes used in this protocol are for 75 cm² flask; 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 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).
4. 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.
5. Add 6.0 to 8.0 mL of complete growth medium and aspirate cells by gently pipetting.
6. Add appropriate aliquots of the cell suspension to new culture vessels. For plates (50mm) use an inoculum of 3 X 10⁵ cells per plate and subculture every 3 days. For 75 cm² flasks use 4 X 10⁵ cells per flask and subculture every 3 days. 
7. Incubate cultures at 37°C.

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Nucleotide (GenBank) : X85983 M.musculus mRNA for carnitine acetyltransferase.

Nucleotide (GenBank) : U85711 Mus musculus phospholipase C delta-1 mRNA, complete cds.

Nucleotide (GenBank) : U85713 Mus musculus phospholipase C-beta-1b mRNA, complete cds.

Nucleotide (GenBank) : U85714 Mus musculus phospholipase C-beta-1b' mRNA, complete cds.