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Complete Growth Medium
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The base medium for this cell line is ATCC-formulated RPMI-1640 Medium, Catalog No. 30-2001. To make the complete growth medium, add the following components to the base medium: 0.2 Units/ml bovine insulin; fetal bovine serum to a final concentration of 10%.
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Subculturing
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- Remove and discard culture medium.
- Briefly rinse the cell layer with either Hanks' Balanced Salt Solution (HBSS) or 0.25% (w/v) Trypsin - 0.53 mM EDTA solution to remove all traces of serum that contains trypsin inhibitor.
- Add 1.0 to 2.0 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 10 to 15 mL of complete growth medium and aspirate cells by gently pipetting.
- Add appropriate aliquots of the cell suspension to new culture vessels. An inoculum of 2 X 104 to 4 X 104 viable cells/cm2 is recommended.
- Incubate cultures at 37C. We recommend that you maintain cultures at a cell concentration between 3 X 104 and 2 X 105 cells/cm2.
Subcultivation Ratio: 1:2 to 1:3 is recommended
Medium Renewal: every 2 to 3 days
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Cryopreservation
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Freeze medium: culture medium, 95%; DMSO, 5% 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°C
Duration: The depositor states that supplementing the growth medium with insulin is optional.
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