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The Aedes albopictus [ATC-15] cell line was established from a pool of several hundred freshly hatched larvae.

1. Remove culture medium to a centrifuge tube.
2. Add 2.0 to 3.0 ml of 0.25%Trypsin-0.53mM EDTA solution to flask and observe cells under an inverted microscope until cell layer is dispersed (usually with 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.
3. Add 6.0 to 8.0 mL of complete growth medium and aspirate cells by gently pipetting.
4. To remove trypsin-EDTA solution, transfer cell suspension to the centrifuge tube and spin at approximately 125 x g for 5 to 10 minutes.
5. Discard supernatant and resuspend cells in fresh growth medium. Add appropriate aliquots of cell suspension to new culture vessels. An inoculum of 7 to 8 x 10³ viable cells/cm² is recommended. Maintain cultures at a cell density between 1 x 10⁴ and 1 x 10⁵ cells/cm².
6. Incubate cultures at 28°C in 95% air, 5% CO₂

Note: For more information on enzymatic dissociation and subculturing of cell lines consult Chapter 10 in Culture of Animal Cells, a Manual of Basic Technique by R. Ian Freshney, 3rd edition, published by Alan R. Liss, N.Y., 1994.

Singh KRP. Cell cultures derived from larvae of Aedes albopictus (Skuse) and Aedes aegypti (L.). Curr. Sci. 36: 506-508, 1967.

Singh KRP, Paul SD. Multiplication of arboviruses in cell lines from from Aedes albopictus and Aedes aegypti. Curr. Sci. 37: 65-67, 1968.

Singh KR. Propagation of arboviruses in Singh's Aedes cell lines. I. Growth of arboviruses in Aedes albopictus and A. aegypti cell lines. Curr. Top. Microbiol. Immunol. 55: 127-133, 1971. PubMed: 5142320

Mitsuhashi J, Maramorosch K. Leafhopper tissue culture: Embryonic, nymphal and imaginal tissues from aseptic insects. Contrib. Boyce Thompson Inst. 22: 435-460, 1964.

Buckley SM. Susceptibility of the Aedes albopictus and A. aegypti cell lines to infection with arboviruses. Proc. Soc. Exp. Biol. Med. 131: 625-630, 1969. PubMed: 5787147

Yunker CE, Cory J. Colorado tick fever virus: growth in a mosquito cell line. J. Virol. 3: 631-632, 1969. PubMed: 5798247

tissue from freshly hatched larvae

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.