Volumes used in this protocol are for 75 cm2 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 0.25% (w/v) Trypsin-0.53 mM EDTA solution to remove all traces of serum that contains trypsin inhibitor.
- 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.
- Add 6.0 to 8.0 mL of complete growth medium and aspirate cells by pipetting gently.
- Add appropriate aliquots of cell suspension to new culture vessels.
- Incubate culture vessels at 37°C.
Protocol for Wnt-3A Conditioned Medium:
- Split the cells 1:10 in 10 mL culture medium (without G418 if conditioned medium is to be used with a cell line sensitive to G418) in 10 cm tissue culture dishes or T-75 flasks and let the cells grow for 4 days (approximately to confluency).
- Take off the medium and sterile filter. This is the first batch of medium.
- Add 10 mL fresh culture medium and culture for another 3 days.
- Take off the medium and sterile filter. This is the second batch of medium. Discard the cells, because they will be overgrown.
- Mix the first batch and second batch of medium 1:1. This is the Wnt-3A conditioned medium. It is stable at 4°C and can be frozen.
Subcultivation Ratio: A subcultivation ratio of 1:4 to 1:10 is recommended.
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.
Freeze medium: Complete growth medium supplemented with 5% (v/v) DMSO
Storage temperature: liquid nitrogen vapor phase
Atmosphere: air, 95%; carbon dioxide (CO2), 5%