青青草原av-午夜视频免费看-免费久久久-中国av片-欧美一级欧美三级在线观看-火影忍者羞羞漫画-成av人片在线观看www-国产日本亚洲-欧美视频在线免费-日本韩国在线-在线日韩中文字幕-国产成人三级在线播放-久久福利在线-老司机免费精品视频-男人操女人逼逼视频-av大片免费-欧美精品第二页-操校花视频-欧美插插视频-优优色综合

1. Remove and discard culture medium.
2. 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.
3. Add 6.0 to 8.0 mL of complete growth medium and aspirate cells by gently pipetting.
4. Add appropriate aliquots of the cell suspension to new Bovine Collagen type l coated flasks. (0.03 mg/mL)
5. Incubate cultures at 37°C. Myotubes form at confluency. Differentiation is improved by reducing the concentration of both sera to 2% each

Subcultivation Ratio: 1:2 to 1:4
Medium Renewal: Every 2 to 3 days

Complete growth medium described above supplemented with 5% (v/v) DMSO.  Cell culture tested DMSO is available as ATCC Catalog No. 4-X.

Christian CN, et al. Synapse formation between two clonal cell lines. Science 196: 995-998, 1977. PubMed: 193191

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.