Subculturing
Volumes used in this protocol are for 75 cm
2 flask; proportionally reduce or increase amount of dissociation medium for culture vessels of other sizes.
- Remove and discard culture medium.
- Briefly rinse the cell layer with PBS to remove all traces of serum which contains trypsin inhibitor.
- Remove the PBS completely and add 2.0 to 3.0 ml of 0.25% (w/v) Trypsin-053mM 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.
- Add 6.0 to 8.0 mL of complete growth medium containing 10% FBS and aspirate cells by gently pipetting.
- To remove trypsin-EDTA solution, transfer cell suspension to centrifuge tube and spin at approximately 125 x g for 5 to 10 minutes.
- Discard supernatant and resuspend cells in fresh culture medium. Add appropriate aliquots of cell suspension to new culture vessels.
- Place culture vessels in incubators at 37°C.
Subcultivation Ratio: 1:3 to 1:6.
Medium Renewal: Every 2 to 3 days.
Note: For more information on enzymatic dissociation and subculturing of cell lines consult Chapter 10 in Culture of Animal Cells, a manual of Basic Technique by R. Ian Freshney, 3rd edition, published by Alan R. Liss, N.Y., 1994
