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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 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 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.
7. Cells will take about 3 days to attach fully. Cells attach as small islands which begin to flatten and spread into patchy epithelial morphology by day 3 or 4.

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Chick WL, et al. A transplantable insulinoma in the rat. Proc. Natl. Acad. Sci. USA 74: 628-632, 1977. PubMed: 191819

Bhathena SJ, et al. Insulin, glucagon, and somatostatin receptors on cultured cells and clones from rat islet cell tumor. Diabetes 31: 521-531, 1982. PubMed: 6295859

Gazdar AF, et al. Continuous, clonal, insulin- and somatostatin-secreting cell lines established from a transplantable rat islet cell tumor. Proc. Natl. Acad. Sci. USA 77: 3519-3523, 1980. PubMed: 6106192

Oie HK, et al. Clonal analysis of insulin and somatostatin secretion and L-dopa decarboxylase expression by a rat islet cell tumor. Endocrinology 112: 1070-1075, 1983. PubMed: 6129963