1. Defrost the cells quickly, then transfer the cell suspension to a sterile 15-mL tube containing prewarmed media (approx 10 mL), and then spin at 1000g for 5 min at room temperature.
2. Aspirate the supernatant, and then add 1 drop of PBS.
3. Resuspend the cells by either flicking the bottom of the tube or by gently pipeting up and down.
4. Add few milliliters of media to the tube, and again gently pipet up and down to dissociate the cells.
5. Plate the cell suspension onto a 6-cm plate, and place in an incubator.
6. Change the media after 8 h or the following morning (if defrosting is carried out late in the evening), and then daily until passaging is required.
7. When the cells reach approx 70% confluence (usually taking 2 d), they are ready to passage.
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