Complete Growth Medium
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DMEM (ATCC 30-2002), 10% FBS (ATCC 30-2020), 3µg/mL Blasticidin (Life Technologies A11139-03)
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Subculturing
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Volumes used in this protocol are for 75 cm2 flasks; proportionally reduce or increase amount of dissociation solutions for culture vessels of other sizes.
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Remove and discard spent medium.
- Briefly rinse with dPBS (ATCC 30-2200), 1 mL per 25 cm2 and discard rinse solution.
- Add 0.25% trypsin (ATCC 30-2101), 1mL per 25 cm2. Place at 37°C for 2-3 minutes, until 90% of the cells have detached.
- Rapt flask gently to ensure cells are detached. Add complete growth media, 1 mL per 25 cm2 to neutralize trypsin.
- Centrifuge cells at 200 x g for 5 min at room temperature.
- Remove supernatant. Resuspend pellet in 5 to 10 mL Complete Growth Medium.
- Count cells, and seed 5.0 x 103 to 8.0 x 103 viable cells/cm2 to new culture vessels.
Subculture every 2-3 days at 8000 cells/cm2
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Cryopreservation
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Serum Free Cell Freezing Media (ATCC® 30-2600)
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Culture Conditions
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Temperature: 37°C
Atmosphere: air, 95%; carbon dioxide (CO2), 5%
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