International Journal of Cell Cloning, Vol 8, 184-195, Copyright © 1990 by AlphaMed Press
Further enrichment and analysis of rat CFU-s
KF McCarthy and ML Hale
Radiation Biochemistry Department, Armed Forces Radiobiology Research Institute, Bethesda, Maryland 20814-5145.
Using the monoclonal antibody W3/13, which recognizes a determinant
expressed on a sialoglycoprotein, rat marrow cells with the phenotype Thy-1
antigen upper 20% positive (Ox720) and high molecular weight leukocyte
common antigen negative (Ox22-) were separated into W3/13 dim (W3/13d) and
W3/13 bright (W3/13b) subpopulations by single-laser cell sorting. The
spleen colony-forming unit (CFU-s) was found in the W3/13d fraction. A
468-fold enrichment of CFU-s was achieved. Only 20% of the Ox720, Ox22-,
and W3/13d cells were in the S phase of the cell cycle as compared to 56%
of Ox720, Ox22-, and W3/13b cells. Using Indo-1, it was not possible to
demonstrate increases in cytosolic Ca++ levels within the enriched CFU-s
population by colony-stimulating factors (CSFs) or interleukins 1, 2, and
3. However, challenge with the Ca++ ionophore, ionomycin, demonstrated
apparent heterogeneity of intracellular Ca++ management within the enriched
CFU-s population. The source of this heterogeneity is not known. Only a
12-day CFU-s was detected in the rat, and it was predominantly, but not
exclusively, a Rhodamine 123 (Rh123) dull cell.
