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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 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. An inoculum of 2 x 10³ to 1 x 10⁴ viable cells/cm² is recommended. Maintain cultures at a cell concentration between 4 x 10³ and 2 x 10⁴ cells/cm².
6. Incubate cultures at 37°C.

Subcultivation Ratio: 1:3 to 1:6
Medium Renewal: Every 2 to 3 days

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.

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

Lemischka IR. Nucleic acids encoding hencatoporetic stem cell receptor flk-2. US Patent 5,185,438 dated Feb 9 1993

Lemischka IR. Nucleic acids encoding fragments of hematopoietic stem cell receptor flk-2. US Patent 5,270,458 dated Dec 14 1993

Lemischka IR. Nucleic acids encoding hematopoietic stem cells receptors flk-1. US Patent 5,283,354 dated Feb 1 1994

Lemischka IR. Tyrosine kinase receptor flk-2 and fragments thereof. US Patent 5,367,057 dated Nov 22 1994

Lemischka IR. Tyrosine kinase receptor human flk-2-specific antibodies. US Patent 5,548,065 dated Aug 20 1996

Lemischka IR. Nucleic acids encoding soluble human FLK-2 extracellular domain. US Patent 5,621,090 dated Apr 15 1997

Lemischka IR. Antibodies against tyrosine kinase receptor flk-1. US Patent 5,747,651 dated May 5 1998

Lemischka IR. Method for isolating stem cells expressing flk-1 receptors. US Patent 5,912,133 dated Jun 15 1999

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.

Biosafety in Microbiological and Biomedical Laboratories, 5th ed. HHS. U.S. Department of Health and Human Services, Centers for Disease Control and Prevention. Washington DC: U.S. Government Printing Office; 2007. The entire text is available online.