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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 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 37C to facilitate dispersal.
4. Add 2.0 to 3.0 mL of complete growth medium and aspirate cells by gently pipetting
5. Resuspend the cell pellet in fresh growth medium. Add appropriate aliquots of the cell suspension to new culture vessels.
6. Incubate cultures at 37°C.

Hull RN, Huseby RM. Enhanced production of plasminogen activator. US Patent 3,904,480 dated Sep 9 1975

Perantoni A, Berman JJ. Properties of Wilms' tumor line (TuWi) and pig kidney line (LLC-PK1) typical of normal kidney tubular epithelium. In Vitro 15: 446-454, 1979. PubMed: 225262

Loffler S, et al. CD9, a tetraspan transmembrane protein, renders cells susceptible to canine distemper virus. J. Virol. 71: 42-49, 1997. PubMed: 8985321