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1. Remove and discard culture medium.
2. Briefly rinse the cell layer with 0.05% (w/v) Trypsin-0.53mM 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 allow flasks to set at room temperature for 3 to 5 minutes.
4. Tap flask and cells will slide off bottom of the flask.
   Note: Cells come off in small clumps and are difficult to break up into single cells without greatly reducing cell viability.
5. Add 6.0 to 8.0 mL of complete growth medium and aspirate cells by gently pipetting. 
6. Add appropriate aliquots of the cell suspension to new culture vessels.

7. Incubate cultures at 30°C.

Subcultivation Ratio: 1:4 to 1:8
Medium Renewal: Every 2 to 3 days

Note: For more information on enzymatic dissociation and subculturing of cell lines consult Chapter 10 in Culture of Animal Cells, a Manual of Basic Technique by R. Ian Freshney, 3rd edition, published by Alan R. Liss, N.Y., 1994.

Complete growth medium described above supplemented with 5% (v/v) DMSO.  Cell culture tested DMSO is available as ATCC Catalog No. 4-X.

Celander M, et al. Cytochromes P450 (CYP) in the Poeciliopsis lucida hepatocellular carcinoma cell line (PLHC-1): dose- and time-dependent glucocorticoid potentiation of CYP1A induction without induction of CYP3A. Arch. Biochem. Biophys. 329: 113-122, 1996. PubMed: 8619627

Huuskonen SE, et al. A fish hepatoma cell line (PLHC-1) as a tool to study cytotoxicity and CYP1A induction properties of cellulose and wood chip extracts. Chemosphere 36: 2921-2932, 1998. PubMed: 9734273

Schultz ME, Schultz RJ. Transplantable chemically-induced liver tumors in the viviparous fish Poeciliopsis. Exp. Mol. Pathol. 42: 320-330, 1985. PubMed: 2987024

Tocher DR, et al. Occurrence of 22:3n-9 and 22:4n-9 in the lipids of the topminnow (Poeciliopsis lucida) hepatic tumor cell line, PLHC-1. Lipids 30: 555-565, 1995. PubMed: 7651084

Fent K. Ecotoxicology of organotin compounds. Crit. Rev. Toxicol. 26: 1-117, 1996. PubMed: 8833456

Babich H, et al. In vitro cytotoxicity studies with the fish hepatoma cell line, PLHC-1 (Poeciliopsis lucida). Ecotoxicol. Environ. Saf. 21: 327-336, 1991. PubMed: 1868789

Ryan JA, Hightower LE. Evaluation of heavy-metal ion toxicity in fish cells using a combined stress protein and cytotoxicity assay. Environ. Tox. Chem. 13: 1231-1240, 1994.

Bruschweiler BJ, et al. Inhibitory effects of heavy metals on cytochrome P4501A induction in permanent fish hepatoma cells. Arch. Environ. Contam. Toxicol. 31: 475-482, 1996. PubMed: 8975819

Bruschweiler BJ, et al. Cytotoxicity in vitro of organotin compounds to fish hepatoma cells PLHC-1 (Poecilopsis lucida). Aquat. Toxicol. 32: 143-160, 1995.

Hahn ME, et al. Cytochrome P4501A induction and inhibition by 3,3',4,4'-tetrachlorobiphenyl in an Ah receptor-containing fish hepatoma cell line (PLHC-1). Aquat. Toxicol. 26: 185-208, 1993.

Bruschweiler BJ, et al. Inhibition of cytochrome P4501A by organotins in fish hepatoma cells PCHC-1. Environ. Tox. Chem. 15: 728-735, 1996.

Bruschweiler BJ, et al. An ELISA asay for cytochrome P4501A in fish liver cells. Environ. Tox. Chem. 15: 592-596, 1996.

Hahn ME, et al. Rapid assessment of induced cytochrome P4501A protein and catalytic activity in fish hepatoma cells grown in multiwell plates: response to TCDD, TCDF, and two planar PCBS. Environ. Tox. Chem. 15: 582-591, 1996.

Hay, R. J., Caputo, J. L., and Macy, M. L., Eds. (1992), ATCC Quality Control Methods for Cell Lines. 2nd edition, Published by ATCC.

Caputo, J. L., Biosafety procedures in cell culture. J. Tissue Culture Methods 11:223-227, 1988.

Fleming, D.O., Richardson, J. H., Tulis, J.J. and Vesley, D., (1995) Laboratory Safety: Principles and Practice. Second edition, ASM press, Washington, DC.

Biosafety in Microbiological and Biomedical Laboratories, 5th ed. HHS. U.S. Department of Health and Human Services, Centers for Disease Control and Prevention. Washington DC: U.S. Government Printing Office; 2007. The entire text is available online.