SoNar-low FL hematopoietic cells exhibit similar glycolytic but enhanced mitochondrial activity compared with SoNar-high cells. (A) Fluorescence-activated cell sorting–purified CD45⁺ SoNar-high and -low FL hematopoietic cells were evaluated for the ratios of SoNar fluorescence with excitation at 405 and 488 nm by confocal microscopy, and representative images are shown. Scale bar, 10 μm. (B) Quantification of the ratios of SoNar fluorescence (F405/F488 nm) in panel A as measured by confocal microscopy. A total of 50 to 60 CD45⁺ SoNar-high and -low FL hematopoietic cells were analyzed (n = 3). (C-D) CD45⁺ SoNar-high (C) and -low FL (D) hematopoietic cells were incubated with PBS, pyruvate, oxamate, AOA, or rotenone, followed by the measurement of the ratios of SoNar fluorescence at indicated time points. A total of 20 to 34 CD45⁺ SoNar-high and -low FL hematopoietic cells were analyzed (n = 3). (E-F) Quantification of the ratios of SoNar fluorescence in CD45⁺ SoNar-high (E) and -low (F) FL hematopoietic cells upon PBS, pyruvate, oxamate, AOA or rotenone treatment (n = 3). (G) CD45⁺ SoNar-high and -low FL hematopoietic cells were cultured with carbon 13–labeled glucose for 2 hours, followed by the determination of intermetabolites derived from glycolysis or TCA cycle (n = 3). (H) OCR levels were examined in CD45⁺ SoNar-high and -low FL hematopoietic cells using a Seahorse XF96 analyzer (n = 3). (I) Quantification of the mean fluorescence intensities (MFIs) of mitochondrial membrane potential in CD45⁺ SoNar-high and -low FL hematopoietic cells by staining with TMRE probe (n = 5). (J) ATP levels were examined in CD45⁺ SoNar-high and -low FL hematopoietic cells (n = 3). (K) mtDNA copies were measured in CD45⁺ SoNar-high and -low FL hematopoietic cells by PCR (n = 3). (L) Plots of the extracellular acidification rate (ECAR) as a parameter of time were examined in CD45⁺ SoNar-high and -low FL hematopoietic cells using a Seahorse XF96 extracellular flux analyzer (n = 3). (M) Extracellular lactate production was measured in the supernatant of the cultured CD45⁺ SoNar-high and -low FL hematopoietic cells 1 hour after culture (n = 3). Data are represented as mean ± standard error of the mean. Student 2-tailed unpaired t test (B,I,J,K,M), 1-way analysis of variance (ANOVA) with Tukey’s multiple comparison test (E,F), and 2-way ANOVA with Sidak’s multiple comparison test (G) were used for the comparison of statistical significance. See also supplemental Figure 3. *P < .05, **P < .01, ***P < .001.