Functional HSCs are enriched in SoNar-low FL hematopoietic cells. (A-B) Representative flow cytometric analyses (A) and quantification (B) of frequencies of long-term HSCs (Lin⁻Sca-1⁺c-Kit⁺CD150⁺CD48⁻) in SoNar-high, -mid, and -low FL hematopoietic cells (n = 5). (C) Cell numbers of immunophenotypic HSCs were counted in SoNar-high, -mid, and -low and total FL hematopoietic cells (n = 5). (D-E) Representative images of the ratios of SoNar fluorescence of FL-HSCs (D); a total of 208 FL-HSCs were examined, and quantitative data are shown (E; n = 3). Scale bar, 10 μm. (F) Quantification of the ratios of SoNar fluorescence (F405/F488 nm) in total FL hematopoietic cells and FL-HSCs as measured by flow cytometric analysis (n = 3). (G) Quantification of ratios of SoNar fluorescence (F405/F488 nm) in total CD45⁺ FL hematopoietic cells and FL-HSCs (n = 5). (H-I) ATP levels and mtDNA copies were examined in total CD45⁺ FL hematopoietic cells and FL-HSCs, respectively (n = 3). (J) Representative flow cytometric plots of the frequencies of repopulated cells in the peripheral blood of recipients transplanted with CD45⁺ SoNar-high or -low FL donor cells (CD45.2) or competitive cells (CD45.1). (K) Repopulated donor cells were analyzed in recipients transplanted with CD45⁺ SoNar-high or -low FL donor cells at 4, 8, and 16 weeks posttransplantation (n = 5). (L) Multilineage contribution of donor cells in the primary recipients at 16 weeks posttransplantation in panel K (n = 5). (M) Secondary transplantation was performed with donor BM cells of primary recipients in panel L. Percentages of repopulated cells were analyzed at 4, 8, and 16 weeks posttransplantation (n = 5). (N) Multilineage contribution of donor cells in the secondary recipients at 16 weeks posttransplantation (n = 5). Repopulated donor cells were analyzed in recipients transplanted with CD45⁺ SoNar-high or -low FL donor cells at 4, 8, and 16 weeks posttransplantation (n = 5). (O-P) Repopulated donor cells were analyzed in recipients transplanted with SoNar-high or -low FL-HSCs cells at 4, 8, and 16 weeks after either primary (O) or secondary transplantation (P; n = 5). (Q) Different cell numbers of CD45⁺ SoNar-high and -low FL donor cells were injected into lethally irradiated recipients, and the competitive repopulating units (CRUs) were determined using L-Calc software. Data are represented as mean ± standard error of the mean. Student 2-tailed unpaired t test (G-I), 1-way analysis of variance (ANOVA) with Tukey’s multiple comparison test (B-C), and 2-way ANOVA with Sidak’s multiple comparison test (K-P) were used for the comparison of statistical significance. See also supplemental Figure 4. *P < .05, **P < .01, ***P < .001.