http://www.cdc.gov/biosafety/publications/bmbl5/index.htm.

The STO cell line was transfected with a G418-resistance cassette, RV4.0 PubMed: 2822260 and a LIF expression construct PubMed: 3143916. Inclusion of G418 in the medium is not necessary for normal cell growth.xa0Note: ATCC tested that this cell line is resistant to:xa0G 418 (neomycin): 350 µg/mL.

Volumes used in this protocol are for 75 cm² flask; proportionally reduce or increase amount of dissociation medium for culture vessels of other sizes.

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 until cell layer is dispersed (usually within 5 to 10 minutes).xa0Note: To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach.xa0 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 gently pipetting.xa0
5. Add appropriate aliquots of the cell suspension to new culture vessels. An inoculum of 1 X 10⁴ viable cells/cm² is recommended.
6. Incubate cultures at 37°C.

Interval: every 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, 5th edition, published by Wiley-Liss, N.Y., 2005.xa0 xa0

McMahon AP, Bradley A. The Wnt-1 (int-1) proto-oncogene is required for development of a large region of the mouse brain. Cell. 62(6): 1073-1085, 1990. PubMed: 2205396

Takahashi K, et al. Induction of pluripotent stem cells from fibroblast cultures. Nat. Protoc. 2(12): 3081-3089, 2007. PubMed: 18079707

Thomas KR, Capecchi MR. Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells. Cell 51(3): 503-512, 1987. PubMed: 2822260

Williams RL, et al. Myeloid leukaemia inhibitory factor maintains the developmental potential of embryonic stem cells. Nature 336: 684-687, 1988. PubMed: 3143916

Okita K, et al, Generation of germline-competent induced pluripotent stem cells. Nature 448(7151): 313-317, 2007. PubMed: 17554338

Hay RJ, Caputo JL, Macy, ML, Eds. (1992) ATCC Quality Control Methods for Cell Lines. 2nd edition, Published by ATCC.

Fleming, D.O., Richardson, J. H., Tulis, J.J. and Vesley, D., (1995) Laboratory Safety: Principles and Practice. Second edition, ASM press, Washington, DC.