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Volumes used in this protocol are for 75 cm² flask; proportionally reduce or increase amount of dissociation medium for culture vessels of other sizes.
Note: The cells are loosely adherent and will detach at room temperature.

1. Remove culture medium to a centrifuge tube.
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 with 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. To remove trypsin-EDTA solution, transfer cell suspension to the centrifuge tube with the medium and cells from step #1 and spin at approximately 125 x g for 5 to 10 minutes.
6. Discard supernatant and resuspend cells in fresh growth medium. Add appropriate aliquots of cell suspension to new culture vessels.
   An inoculum of 5 x 10³ to 5 x 10⁴ viable cells/mL is recommended.
7. Place culture vessels in incubators at 37°C.

Note:For more information on enzymatic dissociation and subculturing of cell lines consult Chapter 13 in Culture of Animal Cells, A Manual Of Basic Technique by R. Ian Freshney, 5th edition, published by Wiley-Liss, N.Y., 2005.

Temperature: 37°C
Atmosphere: Air, 95%; Carbon dioxide (CO₂), 5%

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Magistrelli G, et al. Rapid, simple and high yield production of recombinant proteins in mammalian cells using a versatile episomal system. Protein Expression and Purification: 72(2): 209-216, 2010. PubMed: 20399863

Dobi A, et al. Mammalian expression cloning of nucleic acid binding proteins by agarose thin-layer gelshift clone selection. Biotechniques: 33(4): 868-872, 2002. PubMed: 12398195