The resulting cells were co-transfected with pMSVNeo and selected for growth in medium containing 0.5 mg/ml G418.

1. Remove and discard culture medium.
2. Briefly rinse the cell layer with with Ca++/Mg++ free Dulbeccos phosphate-buffered saline (D-PBS) or 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 with 5 to 10 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. Add appropriate aliquots of cell suspension to new culture vessels.
6. Place culture vessels in incubators at 37°C.

Subcultivation Ratio: A subcultivation ratio of 1:4 to 1:8 is recommended
Medium Renewal: At the time of subcultivation

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Fleming, D.O., Richardson, J. H., Tulis, J.J. and Vesley, D., (1995) Laboratory Safety: Principles and Practice. Second edition, ASM press, Washington, DC.