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

1. Remove culture medium, which contains suspended cells, to a centrifuge tube.
2. Briefly rinse the cell layer 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. xa0Add 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 10 minutes).

Note: To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach.xa0 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.xa0
5. To remove trypsin-EDTA solution, transfer cell suspension to the centrifuge tube containing the medium and cells from step #1 and centrifuge at approximately 125 x g for 5 to 10 minutes.
6. Discard supernatant and resuspend cells in fresh growth medium.xa0 Add appropriate aliquots of cell suspension to new culture vessels.
7. Incubate cultures at 37°C.

Subcultivation Ratio: A subcultivation ratio of 1:2 to 1:3 is recommended

Medium Renewal: Every 2 to 3 days.xa0

NCI-Navy Medical Oncology Branch Cell Line Supplement. J. Cell. Biochem. suppl. 24: 1996.