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Complete Growth Medium
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A 4:1 mixture of Dulbecco's medium and Medium 199 with supplements as follows :
4 parts of Dulbecco's Modified Eagle's Medium containing 4 mM L-glutamine, 4.5 g/L glucose and 1.5 g/L sodium bicarbonate
1 part of Medium 199
Supplemented with:
0.01 mg/ml hygromycin B
10% fetal bovine serum
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Subculturing
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Volumes are given for a 75 cm 2 flask. Recommended use of Corning ® T-75 flasks (catalog #430641). Increase or decrease the amount of dissociation medium needed proportionally for culture vessels of other sizes.
- Remove and discard culture medium.
- Add 3.0 to 5.0 mL of 0.25% trypsin-0.53 mM EDTA solution to the flask and observe cells under an inverted microscope until the cell layer is dispersed (usually within 5 to 15 minutes).
Note: To avoid clumping do not hit or shake 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 10.0 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 3 x 103 to 5 x 103 viable cells/cm2 is recommended. Maintain cultures at a cell concentration between 8 x 103 and 1 x 104 cells/cm2.
- Subcultivation ratio: 1:2 to 1:3 twice weekly
- Incubate cultures at 37°C.
Subcultivation Ratio: 1:2 to 1:3 twice weekly
Medium Renewal: every 2 to 3 days
Note: For more information on enzymatic dissociation and subculturing of cell lines consult Chapter 13 in Culture Of Animal Cells: A Manual of Basic Technique by R. Ian Freshney, 5th edition, published by Wiley-Liss, N.Y., 2005.
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Cryopreservation
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Freeze medium: culture medium, 30%; fetal bovine serum, 60%; DMSO, 10% 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
Growth Conditions: Subculture when cell concentration reaches between 8 X 103 and 1 X 104 cells/cm2.
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