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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.

NOTE: Set up in advance flasks with irradiated fibroblast feeder layers (e.g., X-irradiated STO cells, ATCC 56-X™)

1. Remove and discard culture medium.
2. Briefly rinse the cell layer with 0.25% (w/v) Trypsin- 0.53 mM EDTA solution to remove all traces of serum, which contains trypsin inhibitor.
3. 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 10 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.
4. Add 6.0 to 8.0 mL of complete growth medium and aspirate cells by gently pipetting. 
5. To remove trypsin-EDTA solution, transfer cell suspension to a centrifuge tube and spin at approximately 125 x g for 5 to10 minutes.
6. Discard supernatant and resuspend cells in fresh growth medium. Seed cultures with 8 x 10⁴ cells/cm² in flasks with feeder layer.
7. Incubate cultures at 37°C. Subculture every 3 days to maintain in an undifferentiated proliferative state.

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