hen it will take the culture one cell division to reach confluency again; if a quarter of the cells are transferred then it will take 2 cell divisions, and so on. If a culture divides once every 30 h or so, then in one week it will undergo approximately 5 cell divisions. A split ratio of 1:32 (1:25) should therefore be appropriate for the cells to reach confluency in about one week. In step 1, resuspend the cells in 8 ml medium, and transfer 0.25 ml to the new cell culture vessel.

3. Incubate cells under their usual growth conditions.

Reference
1. Freshney, R.I. (1993) Culture of Animal Cells: a Manual of Basic Technique. 3rd ed. New York: Wiley-Liss.

2. Ausubel, F.M. et al., eds. (1991) Current Protocols in Molecular Biology. New York: Wiley Interscience.

3. Spector, D., Goldman, R.R., and Leinwand, L.A., eds. (1998) Cells: a Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.

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