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Volumes are for a T-75 flask; Adjust accordingly

1. Remove and discard the cell culture medium from the flask.
2. Rinse the cell monolayer with Dulbecco’s PBS without calcium or magnesium and remove.
3. Add 3 to 4 ml of the trypsin-EDTA solution, rotate flask to rinse cell monolayer, remove trypsin solution, and incubate at 37^oC.
4. Once the cells appear to be detached, add 10 ml of complete growth medium with a pipette to the cell suspension to inactivate the trypsin. Gently wash any remaining cells from the growth surface of the   flask. Check the  cells with the microscope to be sure that most (>95%) are single cells. If cell clusters are apparent, continue to disperse the cells with gentle pipetting.
5. Subculture as necessary.
6. Place the flask back into the incubator. Examine the culture the following day to ensure the cells have reattached and are actively growing.
7. Repeat when cells reach confluence.

Pass

Slingluff C, et al. Melanomas with concordant loss of multiple melanocytic differentiation proteins: immune escape that may be overcome by targeting unique or undefined antigens. Cancer Immunol. Immunother. 48:661-672, 2000. PubMed: 10752474

Molhoek K, et al. Human melanoma cytolysis by combined inhibition of mammalian target of rapamycin and Vascular Endothelial Growth Factor/Vascular Endothelial Growth Factor Receptor-2. Cancer Res. 68: (11), 2008. PubMed: 18519701

Molhoek K, et al. Comprehensive analysis of RTK activation in human melanomas reveals autocrine signaling through IGF-1R. Melanoma Res 21(4): 274–284, 2011. PubMed: 21654344