1. Remove and discard culture medium.
2. Briefly rinse the cell layer with Ca++/Mg++ free Dulbeccos phosphate-buffered saline (D-PBS) or 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 at room temperature until cell layer is dispersed (usually within 5 to 10 minutes).
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 28°C without a mixture of CO₂ in air.

Subcultivation Ratio: 1:2
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, 3rd edition, published byxa0Wiley-Liss, N.Y., 2005.

Complete growth medium described above supplemented with 5% (v/v) DMSO.xa0 Cell culture tested DMSO is available as ATCC Catalog No. 4-X.

Kuno G. New cell line. A continuous cell line of a nonhematophagous mosquito, Toxorhynchites amboinensis. In Vitro 16: 915-917, 1980.

Varma MG, Pudney M. The growth and serial passage of cell lines from Aedes aegypti (L.) larvae in different media. J. Med. Entomol. 6: 432-439, 1969. PubMed: 5360492

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.