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 epithelial cell line was derived from the normal bronchial tissue of a 2- to 3-year-old rhesus monkey. The line was initiated in May, 1978 from tissue explants using glass chips.xa0

epidermal growth factor (EGF), expressed

keratin

Cells contain PAS-positive cytoplasmic inclusions.

Volumes used in this protocol are for 75 cm² flask; proportionally reduce or increase amount of dissociation medium for culture vessels of other sizes.xa0

1. Remove and discard culture medium.
2. 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.
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. 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.
6. Incubate cultures at 37°C.

Subcultivation Ratio: A subcultivation ratio of 1:2 to 1:4 is recommended

Medium Renewal: 2 to 3 times per week

Note: The line requires murine epidermal growth factor (EGF) for proliferation, which may be aquired from BD Bioscience (BD354001) or Sigma (E-4127).

Freeze medium: Complete growth medium supplemented with 5% (v/v) DMSO

Storage temperature: liquid nitrogen vapor phase

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

Temperature: 37°C