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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 pipetting gently.
5. Add appropriate aliquots of cell suspension to new culture vessels.
6. Incubate culture vessels at 37°C.
   

Condit RC, Motyczka A. Isolation and preliminary characterization of temperature-sensitive mutants of vaccinia virus. Virology 113: 224-241, 1981. PubMed: 7269240

Brockman WW, Nathans D. The isolation of simian virus 40 variants with specifically altered genomes. Proc. Natl. Acad. Sci. USA 71: 942-946, 1974. PubMed: 4362640

Ensinger MJ. Isolation and genetic characterization of temperature-sensitive mutants of vaccinia virus WR. J. Virol. 43: 778-790, 1982. PubMed: 7143565

BSC40 is a high-temperature tolerant derivative of BS-C-1 established by Brockman and Nathans. BSC40 cells may be grown at temperature from 31C to 40C. The cells are useful for selection and assay of temperature sensitive viruses. They support the growth of vaccinia virus, and can be used as a host for vaccinia virus genetic studies.