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Note: Never allow cultures to become completely confluent before subculture. 

Some important considerations in the handling of 3T3 cells: doubling time is about 18 hours in sparse cultures. The cells reach a saturation density of about 10⁶ cells per 20 cm². In order to maintain this property of high contact inhibition, it is necessary to transfer routinely at only high dilutions, otherwise variants tend to be selected having reduced contact inhibition. Such low density makes culture vessels appear sparse and cell growth sensitive to sub-optimal temperature and media conditions.

1. Remove and discard culture medium.
2. Briefly rinse the cell layer with 0.25% (w/v) Trypsin - 0.53 mM EDTA solution to remove all traces of serum that 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 15 minutes).
   Note: To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. 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.
5. Add appropriate aliquots of the cell suspension to new culture vessels.
6. Incubate cultures at 37°C.

Aaronson SA, Todaro GJ. Development of 3T3-like lines from Balb-c mouse embryo cultures: transformation susceptibility to SV40. J. Cell. Physiol. 72: 141-148, 1968. PubMed: 4301006

Todaro GJ, Aaronson SA. Properties of clonal lines of murine sarcoma virus transformed Balb-3T3 cells. Virology 38: 174-202, 1969. PubMed: 4306523

Aaronson SA, Todaro GJ. Basis for the acquisition of malignant potential by mouse cells cultivated in vitro. Science 162: 1024-1026, 1968. PubMed: 4301647

Jainchill JL, Todaro GJ. Stimulation of cell growth in vitro by serum with and without growth factor. Relation to contact inhibition and viral transformation. Exp. Cell Res. 59: 137-146, 1970. PubMed: 4194429

Thompson SA, et al. COOH-terminal extended recombinant amphiregulin with bioactivity comparable with naturally derived growth factor. J. Biol. Chem. 271: 17927-17931, 1996. PubMed: 8663535

Anderson MT, et al. Simultaneous fluorescence-activated cell sorter analysis of two distinct transcriptional elements within a single cell using engineered green fluorescent proteins. Proc. Natl. Acad. Sci. USA 93: 8508-8511, 1996. PubMed: 8710900

Biological evaluation of medical devices. Part 5: Tests for in vitro cytotoxicity. Sydney, NSW, Australia:Standards Australia;Standards Australia AS ISO 10993.5-2002.

Biological evaluation of medical devices--Part 5: Tests for in vitro cytotoxicity. Geneva (Switzerland):International Organization for Standardization/ANSI;ISO ISO 10993-5:1999.