Biosafety classification is based on U.S. Public Health Service Guidelines, it is the responsibility of the customer to ensure that their facilities comply with biosafety regulations for their own country.

This cell line is insensitive to epsilon toxin from C.perfringens and is a control for MDCK.2 (ATCC CRL-2936)

Cell line was derived by cloning (limited dilution) the parental cell line MDCK (ATCC CCL-34).xa0

E-cadherin (epithelial cell adhesion molecule), expressed

Zona Occludens (ZO-1) (tight junction protein), not expressed

CD29, expressed

CD18, not expressed

fibroblast-specific protein (FSP), not expressed

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

Volumes used in this protocol are for 75xa0cm² 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 Dulbeccos 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 atxa037°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 3 X 10⁴ viable cells/cm² is recommended.
6. Incubate cultures at 37°C. Subculture when the cell concentration is between 8 X 10⁴ and 2 X 10⁵ cells/cm².

Medium renewal: Every 2 to 3 days.

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

liquid nitrogen vapor phase