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Stimulation by other hormones or cAMP is not required for hormone secretion.

Reverse Transcript positive

• fetal bovine serum to a final concentration of 2.5%
• horse serum to a final concentration of 15%

1. Remove and discard culture medium.
2. Briefly rinse the cell layer with 0.25% (w/v) Trypsin-0.53mM EDTA solution diluted 1:10 in PBS to remove all traces of serum which contains trypsin inhibitor.
3. Add 2.0 to 3.0 mL of the diluted 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.
4. Add 6.0 to 8.0 mL of complete growth medium and aspirate cells by gently pipetting.
5. Add appropriate aliquots of the cell suspension to new culture vessels.
6. Incubate cultures at 37°C.

Subcultivation Ratio: A subcultivation ratio of 1:2 to 1:3 is recommended
Medium Renewal: Add fresh medium at least twice per 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.

Shin SI, et al. Studies on interstitial cells in tissue culture. II. Steroid biosynthesis by a clonal line of rat testicular interstitial cells. Endocrinology 82: 614-616, 1968. PubMed: 4868022

Hay, R. J., Caputo, J. L., and Macy, M. L., Eds. (1992), ATCC Quality Control Methods for Cell Lines. 2nd edition, Published by ATCC.

Caputo, J. L., Biosafety procedures in cell culture. J. Tissue Culture Methods 11:223-227, 1988.

Fleming, D.O., Richardson, J. H., Tulis, J.J. and Vesley, D., (1995) Laboratory Safety: Principles and Practice. Second edition, ASM press, Washington, DC.