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This line was derived by inserting a construct containing a disrupted murine Lyt-2 (CD8) gene. 

The Lyt-2 genes was disrupted by insertion of a plasmid containing a neomycin resistance gene into the first exon of the Lyt-2 gene. 

After selection for neomycin resistance and presence of Lyt-2 sequences upstream of the insertion, the 36.5 cell line was established. 

This line has been used to produce mice deficient in expression of CD8.

The cells can be maintained in the undifferentiated state by frequent subculture on feeder layers of mitomycin C treated STO cells (MITC-STO ATCC 56-X.2) (see ATCC 56-X.2, MITC-STO cells).

1. Remove and discard culture medium.
2. Rinse the cell layer with 0.25% (w/v) trypsin-0.03% (w/v) EDTA solution. Remove the trypsin and incubate at 37°C until cells detach (approximately 10 minutes).
3. Add complete growth medium and aspirate cells by gently pipetting.
4. Add appropriate aliquots of the cell suspension to new culture vessels with fresh feeder layer cultures.
5. Incubate cultures at 37°C.

Subcultivation Ratio: 1:3 to 1:6 is recommended
Medium Renewal: 2 to 3 days

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

Mak TW. Mutant mouse lacking CD8 surface marker. US Patent 5,530,178 dated Jun 25 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.