XB-2 cells can undergo a process of terminal differention similar to keratinocytes.

When cultured at low cell density as adherent cells, few cells differentiate.

When placed in suspension culture, however, cultures demonstrate colony forming ability decrease, nuclear pyknosis and other features of keratinocyte differentiation.

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, 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 10 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 with feeder layer X-56.
6. Incubate cultures at 37°C.

Subcultivation Ratio: See subcultivation information above
Medium Renewal: Once per week

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.

Green H, Rheinwald JG. Process for serially culturing keratinocytes. US Patent 4,016,036 dated Apr 5 1977

Morrissey JH, Green H. Differentiation-related death of an established keratinocyte line in suspension culture. J. Cell. Physiol. 97: 469-476, 1978. PubMed: 730781

Stevens LC. The development of transplantable teratocarcinomas from intratesticular grafts of pre- and postimplantation mouse embryos. Dev. Biol. 21: 364-382, 1970. PubMed: 5436899

XB-2 was isolated from a transplantable mouse teratoma (No. 69691) established by L. C. Stevens.

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.