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Adaptation to other types of sera generally results in slow growth with a prolonged lag in growth.

Volumes used in this protocol are for 75 cm² flask; proportionally reduce or increase amount of dissociation medium for culture vessels of other sizes.

1. Remove and discard culture medium.
2. Briefly rinse the cell layer with 0.25% (w/v) Trypsin-0.53mM  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 with 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.
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  centrifuge tube and spin atapproximately 125 x g for 5 to10 minutes.
6. Discard supernatant and resuspend cells in fresh culture medium.  Add appropriate aliquots of cell suspension to new culture vessels. 
7. Place culture vessels in incubators at 37°C.

Subcultivation Ratio: A subcultivation ratio of 1:2 to 1:10 is recommended

American Association of Anatomists. Sixty-ninth annual session; Marquette University School of Medicine, Milwaukee, Wisconsin. Anat. Rec. 124: 490, 1956.

Hull RN, et al. Growth characteristics of monkey kidney cell strains LLC-MK1, LLC-MK2, and LLC-MK2(NCTC-3196) and their utility in virus research. J. Exp. Med. 115: 903-918, 1962. PubMed: 14449901

Hull RN, Tritch OJ. Characterization of cell strains by viral susceptibility. Natl. Cancer Inst. Monogr. 7: 161-172, 1962. PubMed: 14449902