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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.

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Reuber MD. A transplantable bile-secreting hepatocellular carcinoma in the rat. J. Natl. Cancer Inst. 26: 891-899, 1961. PubMed: 13740982

Weinstein IB, et al. Type C virus from cell cultures of chemically induced rat hepatomas. Science 178: 1098-1100, 1972. PubMed: 4343844

Klemm DJ, et al. Adenovirus E1A proteins regulate phosphoenolpyruvate carboxykinase gene transcription through multiple mechanisms. J. Biol. Chem. 271: 8082-8088, 1996. PubMed: 8626493

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Nucleotide (GenBank) : AJ010024 Rattus norvegicus mRNA for Mi-2 autoantigen, partial.