Subculturing
Volumes are given for a 75 cm2 flask. Increase or decrease the amount of dissociation medium needed proportionally for culture vessels of other sizes.
- 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.
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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: An inoculation density of 5 X 104 viable cells per cm2 of flask or dish surface area is recommended
Medium Renewal: 2 to 3 times per week
Note: The tryptose phosphate broth should be prepared fresh every 2 weeks. Fresh glutamine is also very important for the growth of this line.
