Single cell cloning and selection in G-418 of the 3D4 parental cell line resulted in establishment of 3D4/2 (

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 that 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).
   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 the cell suspension to new culture vessels. An inoculum of 5 x 10³ to 7 x 10³ viable cells/cm² is recommended.

6. Incubate cultures at 37°C. Subculture when cell concentration reaches between 3 x 10⁵ and 4 x 10⁵ cells/cm².

Subc*tion Ratio: A subc*tion ratio of 1:6 to 1:8 is recommended

Medium Renewal: Two to three 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.