The variable prevalence of SP cells in different hematopoietic cell populations. Cells were costained with lin⁺ antibodies, Hoechst 33342, and Rhodamine-123 as described in “Materials and methods.” Representative FACS profiles are shown for adult bone marrow (A-C), day-14.5 fetal liver (D-F and P-R), juvenile bone marrow (G-I), day-2 5-FU adult bone marrow (J-L), and adult Sca-1⁺ bone marrow cells cultured for 7 days with SF, IL-11, and flt3-ligand (M-O). The first column of FACS profiles shows the dual-wave Hoechst 33342 emission fluorescence of the total population of PI^- cells for each population examined. The second column of profiles (B-N) shows the subpopulations of SP cells according to their expression of lin⁺ markers and Rhodamine-123 fluorescence. For the analyses of adult bone marrow cells (B-C), Mac1 was included in the lin⁺ antibody cocktail, but in all other analyses Mac1 was omitted. The third column of profiles (C-O) shows the corresponding subsets in the non-SP cells from each of the suspensions tested. The gates shown in the panels in the second and third columns were used to isolate cells for HSC assays. For fetal liver, some cells were also stained with CD45 (which does not stain either hepatic cells or mature erythroid cells), and representative profiles are shown in panels Q-R. Because maturing erythroblasts make up most of the cells in the day 14.5 fetal liver, a large lin⁺CD45^- population is seen (Q-R). The values shown in all of the profiles show the percentages of cells in each subset of the population examined (total cells in the first column, SP cells in the second column, non-SP cells in the third column, and as indicated in panels P-R). Note that in the experiments with cultured adult bone marrow cells, the streptavidin-APC used to visualize the lin⁺ cells gave a very weak staining of these cells and hence a likely underestimation of their numbers (N-O).