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Complete Growth Medium
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RPMI 1640 medium, 90%; heat-inactivated fetal bovine serum, 10%
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Subculturing
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Volumes used in this protocol are for 75 cm2 flask; proportionally reduce or increase amount of dissociation medium for culture vessels of other sizes.
- This is a cell line that grows as both attached and suspended cells. The suspended cells are viable and should not be discarded. Recover the suspended cells by gentle centrifugation (125 x g) for subculture along with the adherent cell population.
- To dissociate adherent cells, 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.
- 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.
- Add appropriate aliquots of the cell suspension to new culture vessels.
- Incubate cultures at 37°C
Subculture Ratio: 1:3 to 1:6
Medium Renewal: 2 to 3 times a week.
Note: For more information on enzymatic dissociation and subculturing of cell lines consult Chapter 10 in Culture of Animal Cells, a manual of Basic Technique by R. Ian Freshney, 3rd edition, published by Alan R. Liss, N.Y., 1994.
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Cryopreservation
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Culture medium, 95%; DMSO, 5%
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