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1. Remove culture medium with floating cells to a centrifuge tube.
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).
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 the centrifuge tube with the medium and cells from step #1 and spin at approximately 125 x g for 5 to 10 minutes.
6. Discard supernatant and resuspend cells in fresh growth medium.  Add appropriate aliquots of cell suspension to new culture vessels. Resuspend cells  at 2 x 10⁵ viable cells/m.
7. Place culture vessels in incubators at 37°C.

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

Reimer CB, et al. Evaluation of thirty-one mouse monoclonal antibodies to human IgG epitopes. Hybridoma 3: 263-275, 1984. PubMed: 6209201

Jefferis R, et al. Evaluation of monoclonal antibodies having specificity for human IgG sub-classes: Results of an IUIS/WHO collaborative study. Immunol. Lett. 10: 223-252, 1985. PubMed: 3899923

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.