国产精品国产精品一区精品国产自在现偷99精品国产在热2019国产拍偷精品网国产精品视频全国免费观看,国产精品v欧美精品v日韩精品青青精品视频国产久久国产精品久久精品国产亚洲精品国产精品国产欧美精品一区二区三区,国产精品第一页国产亚洲精品国产福利国产精品自拍国产精品视频在线观看亚洲国产精品一区二区久久国产精品国产三级国产专不,国产精品视频大陆精大陆国产国语精品2019精品国产品对白在线285年香蕉精品国产高清自在自线隔壁老王国产在线精品在线观看精品国产福利片,国产三级精品三级在专区精品国产自在现偷国产精品一区二区三区国产日韩精品欧美一区喷水亚洲精品国产精品国自产国产在线精品一区二区不卡

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 that 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 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 at approximately 125 x g for 5 to 10 minutes.
6. Discard supernatant and resuspend cells in fresh growth medium with 10% FBS added. Add appropriate aliquots of cell suspension to new culture vessels.
7. Place culture vessels in incubators at 37°C. 24 hours later perform a fluid change with serum free growth medium if desired (Nissley SP, et al.. 1977).

Subcultivation Ratio: A subcultivation ratio of 1:3 to 1:6 is recommended
Medium Renewal: 2 times per week
Note: For 24 hrs after initiating culture from a thawed ampule or after subculturing, grow the cells in culture medium with 10% fetal bovine serum. If desired, after 24 hrs, the cells may be transferred to serum-free medium.

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.

Temperature: 37°C
Atmosphere: Air, 95%; carbon dioxide (CO₂), 5%

Nissley SP, et al. Proliferation of buffalo rat liver cells in serum-free medium does not depend upon multiplication-stimulating activity (MSA). Cell 11: 441-446, 1977. PubMed: 302146

Coon HG, Weiss MC. A quantitative comparison of formation of spontaneous and virus- produced viable hybrids. Proc. Natl. Acad. Sci. USA 62: 852-859, 1969. PubMed: 4308097

Dulak NC, Temin HM. A partially purified polypeptide fraction from rat liver cell conditioned medium with multiplication-stimulating activity for embryo fibroblasts. J. Cell. Physiol. 81: 153-170, 1973. PubMed: 4735141

Dulak NC, Shing YW. Large scale purification and further characterization of a rat liver cell conditioned medium multiplication stimulating activity. J. Cell. Physiol. 90: 127-130, 1977. PubMed: 833209

Schalch DS, et al. Nonsuppressible insulin-like activity (NSILA). I. Development of a new sensitive competitive protein-binding assay for determination of serum levels. J. Clin. Endocrinol. Metab. 46: 664-671, 1978. PubMed: 755052

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.

Nissley SP, et al. Proliferation of buffalo rat liver cells in serum-free medium does not depend upon multiplication-stimulating activity (MSA). Cell 11: 441-446, 1977. PubMed: 302146

Nucleotide (GenBank) : U14746 Rattus norvegicus VHL protein (VHL) mRNA, complete cds.