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Volumes used in this protocol are for 75 cm² flasks; proportionally reduce or increase amount of dissociation medium for culture vessels of other sizes. The suspended cells are viable and can be used to start new cultures.

1. To subculture attached cells, 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.
2. Observe cells under an inverted microscope until cell layer is dispersed. 
   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.
3. Add 6.0 to 8.0 ml of complete growth medium and aspirate cells by gently pipetting. 
4. Add appropriate aliquots of cell suspension to new culture vessels.
5. Place culture vessels in incubators at 37°C.

Subcultivation Ratio: 1:4 to 1:12
Medium Renewal: Two times weekly

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.  

Complete growth medium described above supplemented with 5% (v/v) DMSO.  Cell culture tested DMSO is available as ATCC Catalog No. 4-X.

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