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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.

Martin GR, Evans MJ. Differentiation of clonal lines of teratocarcinoma cells: formation of embryoid bodies in vitro. Proc. Natl. Acad. Sci. USA 72: 1441-1445, 1975. PubMed: 1055416

Teratomas and Differentiation, eds. Michael I. Sherman and David Solter, 13–14. New York: Academic Press, 1975.

Martin GR, Evans MJ. Multiple differentiation of clonal teratocarcinoma stem cells following embryoid body formation in vitro. Cell 6: 467-474, 1975.

Martin GR, et al. The development of cystic embryoid bodies in vitro from clonal teratocarcinoma stem cells. Dev. Biol. 61: 230-244, 1977. PubMed: 590624

The line was derived from a continuous line of SIM mouse embryonic fibroblasts by A. Bernstein at the Ontario Cancer Institute.