| Subculturing |
Volumes used in this protocol are for 75 cm2 flask; proportionally reduce or increase amount of dissociation medium for culture vessels of other sizes.
- Remove and discard culture medium.
- Briefly rinse the cell layer with 0.25% (w/v) Trypsin-0.53 mM EDTA solution containing 0.5% PVP to remove all traces of serum, which contains trypsin inhibitor.
- 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).
- Add 6.0 to 8.0 mL of complete growth medium and aspirate cells by gently pipetting.
- To remove trypsin-EDTA solution, transfer cell suspension to a centrifuge tube and spin at approximately 125 x g for 5 to10 minutes.
- Discard supernatant and resuspend cells in fresh growth medium. Add appropriate aliquots of cell suspension to new culture vessels.
- Place culture vessels in incubators at 28°C.
Subcultivation Ratio: A subcultivation ratio of 1:2 to 1:3 is recommended
Medium Renewal: Twice 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. |
| References |
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.
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.
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