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Complete Growth Medium
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Dulbecco's modified Eagle's medium with 4 mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate and 4.5 g/L glucose and supplemented with 0.1 mM 2-mercaptoethanol, 85%; fetal bovine serum, 15%
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Subculturing
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Protocol: - Remove and discard culture medium.
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Briefly rinse the cell layer with 0.25% (w/v) Trypsin- 0.53 mM EDTA solution to remove all traces of serum that contains trypsin inhibitor.
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Add 2.0 to 3.0 ml of Trypsin-EDTA solution to flask and observe cells under an inverted microscope until cell layer is dispersed (usually within 5 to 15 minutes).
Note: To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach.
Cells that are difficult to detach may be placed at 37°C to facilitate dispersal. -
Add 6.0 to 8.0 ml of complete growth medium and aspirate cells by gently pipetting.
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Add appropriate aliquots of the cell suspension to new culture vessels.
- Incubate cultures at 37°C.
Subcultivation Ratio: A subcultivation ratio of 1:6 to 1:20 is recommended Medium Renewal: Every 2 to 3 days
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Cryopreservation
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Freeze medium: Complete growth medium supplemented with 5% (v/v) DMSO Storage temperature: liquid nitrogen vapor phase
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Culture Conditions
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Atmosphere: air, 95%; carbon dioxide (CO2), 5% Temperature: 37.0°C Growth Conditions: The line should be grown on feeder layers of irradiated (3000 rads) or mitomycin C treated (0.01 mg/ml for 90 minutes) primary mouse embryonic fibroblasts or STO cells (see ATCC CRL-1503 or ATCC 56-X, irradiated STO cells).
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