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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.
6. Incubate cultures at 37°C.

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

Note: For more information on enzymatic dissociation and subculturing of cell lines consult Chapter 13 in Culture Of Animal Cells: A Manual Of Basic Technique by R. Ian Freshney, 5th edition, published by Wiley-Liss, N.Y., 2005.

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

Di Persio JF, et al. Human cell lines that elaborate colony-stimulating activity for the marrow cells of man and other species. Blood 51: 507-519, 1978. PubMed: 304748

DiPersio JF, et al. The fractionation, characterization, and subcellular localization of colony-stimulating activities released by the human monocyte-like cell line, GCT. Blood 56: 717-727, 1980. PubMed: 6968234

Abboud CN, et al. Hydrophobic adsorption chromatography of colony-stimulating activities and erythroid-enhancing activity from the human monocyte-like cell line, GCT. Blood 58: 1148-1154, 1981. PubMed: 6975643

Lichtman MA, Brennan JK. Obtaining human cell lines that elaborate colony stimulating activity for marrow cells of man and other species and methods of preparing same. US Patent 4,135,975 dated Jan 23 1979

Abboud CN, et al. Erythropoietic enhancing activity (EEA) secreted by the human cell line, GCT. J. Supramol. Struct. 13: 199-209, 1980. PubMed: 6972469

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.