The general protocol for freezing cells grown in a standard 10-cm dish at 70% confluency is given below (see Notes 14 and 15):
1. Change media 2-3 h before freezing the cells.
2. Freshly prepare 2X freezing media.
3. Harvest the cells in a 15-mL tube containing DMEM+ after trypsinization (see Note 15).
4. Spin down at 1000g for 5 min at room temperature.
5. Remove the supernatant, and then add one or two drops of DMEM+ to the tube. Shake gently, but thoroughly to disperse the cells.
6. Add an additional DMEM+ medium to a total volume of 1.5 mL, and disperse the cells carefully so that they comprise a single-cell suspension.
7. Add an equal volume (1.5 mL) of 2X freezing medium, and mix by pipeting several times.
8. Quickly aliquot the cell suspension into three vials, and immediately place them in a styrofoam box (this will allow them to cool down gradually). Alternatively, special boxes dedicated to this task can be purchased from a number of manufacturers (for example, Stratagene) (see Note 16).
9. Place the box in a -70°C freezer for 1-2 d, and then transfer the individual cryovials into a liquid nitrogen container for long-term storage (see Note 16).
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