The cells are positive for keratin by immunoperoxidase staining.

xa0

(Note: The L-15 medium formulation was devised for use in a free gas exchange with atmospheric air. A CO2 and air mixture is detrimental to cells when using this medium for cultivation)

Volumes used in this protocol are for 75 cm2 flask; proportionally reduce or increase amount of dissociation medium for culture vessels of other sizes.

Subculture just prior to confluency

1. Remove and discard culture medium.
2. Briefly rinse the cell layer with 0.25% (w/v) Trypsin-0.53mM 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 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.xa0 Cells that are difficult to detach may be placed at 37°C to facilitate dispersal.
4. Add 6.0 to 8.0 mof complete growth medium and aspirate cells by gently pipetting.xa0
5. To remove trypsin-EDTA solution, transfer cell suspension to the centrifuge tube and spin at approximately 125 x g for 5 to10 minutes.
6. Discard supernatant and resuspend cells in fresh growth medium.xa0 Add appropriate aliquots of cell suspension to new culture vessels.
7. Incubate cultures at 37°C without CO_2.

Leibovitz A, et al. Classification of human colorectal adenocarcinoma cell lines. Cancer Res. 36: 4562-4569, 1976. PubMed: 1000501