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Cell line was derived by cloning (limited dilution) the parental cell line MDCK (ATCC CCL-34).

E-cadherin (epithelial cell adhesion molecule), expressed

Zona Occludens (ZO-1) (tight junction protein), expressed

fibroblast-specific protein (FSP), not expressed

cytokeratin (CK1, 4, 5, 6, 8, 10, 13, 18, 19),expressed

sialic receptors: alpha 2,3-galactose (avian)and alpha 2,6-galactose (human); expressed

Zona Occludens (ZO-1) (tight junction protein), expressed,E-cadherin (epithelial cell adhesion molecule), expressed,fibroblast-specific protein (FSP), not expressed ,cytokeratin (CK1, 4, 5, 6, 8, 10, 13, 18, 19),expressed,sialic receptors: alpha 2,3-galactose (avian)and alpha 2,6-galactose (human); expressed

This cell line is susceptible to a wide range of influenza virus and is sensitive to epsilon toxin of C. perfringens.

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

1. Remove and discard culture medium.
2. Briefly rinse the cell layer with Ca++/Mg++ free Dulbecco's phosphate-buffered saline (D-PBS) or 0.25% (w/v) Trypsin - 0.53 mM EDTA solution to remove all traces of serum which contains trypsin inhibitor.
3. Add 1.0 to 2.0 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 1 X 10⁴ to 2 X 10⁴ viable cells/cm² is recommended.
6. Incubate cultures at 37°C. Subculture when the cell concentration is between 7 X 10⁴ and 1 X 10⁵ cells/cm².

Medium renewal: Every 2 to 3 days.

Freeze medium: Complete growth medium, 95%; DMSO, 5%

liquid nitrogen vapor phase

Temperature: 37°C

Atmosphere: air, 95%; carbon dioxide (CO₂), 5%