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1. To subculture attached cells, biefly rinse the cell layer with 0.25% (w/v) Trypsin-0.53 mM EDTA solution to remove all traces of serum, which contains trypsin inhibitor.
2. Observe cells under an inverted microscope until cell layer is dispersed.
   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.
3. Add 6.0 to 8.0 mL of complete growth medium and aspirate cells by gently pipetting.
4. Add appropriate aliquots of the cell suspension to new culture vessels.
5. Incubate cultures at 37°C.

Subcultivation Ratio: 1:4 to 1:12
Medium Renewal: Every 2 to 3 days

Note: For more information on enzymatic dissociation and subculturing of cell lines consult Chapter 13 in Culture of Animal Cells, a Manual of Basic Technique by R. Ian Freshney, 5th edition, published by Wiley-Liss, N.Y., 2005.

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

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Bessho T, et al. Initiation of DNA interstrand cross-link repair in humans: the nucleotide excision repair system makes dual incisions 5" to the cross-linked base and removes a 22- to 28-nucleotide-long damage-free strand. Mol. Cell. Biol. 17: 6822-6830, 1997. PubMed: 9372913

Reardon JT, et al. Isolation and characterization of two human transcription factor IIH (TFIIH)-related complexes: ERCC2/CAKand TFIIH. Proc. Natl. Acad. Sci. USA 93: 6482-6487, 1996. PubMed: 8692841

Thompson LH, et al. Hypersensitivity to mutation and sister-chromatid-exchange induction in CHO cell mutants defective in incising DNA containing UV lesions. Somatic Cell Genet. 8: 759-773, 1982. PubMed: 7163954

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

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