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These cells represent cells obtained from the microenvironment of the liver that support purified mouse and human CD34+CD38- hematopoietic stem/progenitor cells. Delta-like (Dlk1) /preadipocyte factor-1 (pref-1) is preferentially expressed in AFT024 cells and functions as a positive stem cell regulator.  Due to the presence of the temperature sensitive SV40 T antigen, the AFT024 cells must be grown at 33°C. At 33°C, T antigen binds anti-oncogenes and allows cell division. At higher temperatures, such as 37°C, T antigen is inactivated and the cells can no longer divide. 

Volumes used in this protocol are for 75 cm² flask; proportionally reduce or increase amount of dissociation medium for culture vessels of other sizes.

1.  Remove and discard culture medium.
2. Briefly rinse the cell layer with 1X PBS (Ca⁺²/Mg⁺²-free, ATCC SCRR-2201) to remove all traces of serum that contains trypsin inhibitor. 
3. Add 2.0 to 3.0 mL of 0.25% (w/v) Trypsin / 0.53 mM 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 33°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 cell suspension to new culture vessels.

Subcultivation ratio: 1:4 –1:8

6. Place culture vessels in incubators at 33°C.

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.  

Medium Renewal

Two to three times weekly

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Miller JS, et al. Single adult human CD34(+)/Lin-/CD38(-) progenitors give rise to natural killer cells, B-lineage cells, dendritic cells, and myeloid cells. Blood 93: 96-106, 1999. PubMed: 9864151

Moore KA, et al. In vitro maintenance of highly purified, transplantable hematopoietic stem cells. Blood 89: 4337-4347, 1997. PubMed: 9192756

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Punzel M, et al. The myeloid-lymphoid initiating cell (ML-IC) assay assesses the fate of multipotent human progenitors in vitro. Blood 93: 3750-3756, 1999. PubMed: 10339481

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Hay RJ, Caputo JL, Macy, ML, Eds. (1992) ATCC Quality Control Methods for Cell Lines. 2nd edition, Published by ATCC.