Starting+Tissue+Culture+Cells

see the Cell Line Database 2-6-12 or logbook to locate stock of cells.

Current Protocols in Immunology Recommendations

Example Thawing and Plating (B16 Cells)

Thaw and plate B16 cells 1. Rapidly thaw a vial of cryopreserved B16 cells in 37°C water bath until a small ice clump is left in the vial. Prevent “heat shock” to tumor cells. 2. Transfer contents into a 15-ml tube containing 10 ml ice-cold CM. 3. Pellet cells for 10 min at 663 × g (in a Sorvall H-2000B rotor at 1500 rpm), 4°C. Cell pellet should appear light brown to black. 4. Decant supernatant and resuspend cells in 20 ml ice-cold CM. 5. Plate in 175-cm2 tissue culture coated flask and grow in 37°C, 5% CO2 incubator. (Most people in my lab recommend starting with a flask that is smaller than the largest flask (e.g. 75 cm^2 or smaller)

Cells should be subcultured 2 or 3 times before using for an experiment.

Felicia's Advice

To start a new cell line, take one vial, thaw it in the water bath, and then place it into new media in a flask (e.g. 5-7 mL media in the smallest size tissue culture flask). The cell line vials are not reused so just use a whole vial to start a cell line. Felicia likes to start the culture in the morning and let cells adhere for about 4 hr (if the cells don't adhere by this time she concludes that they are bad). Then she will take away the media to remove the DMSO and add fresh media.

See also Freezing Tissue Culture Mammalian Cells