Further enrichment and analysis of rat CFU-s

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.