Figure 1. HSC numbers are normal in... | Rockefeller University Press

Figure 1.

$Figure 1. HSC numbers are normal in steady-state adult BM of Tnfrsf1-dKO mice. (a) In six separate experiments with three mice in each experiment, BM cells from two tibiae and two femurs per mouse were individually isolated from age (8–12 wk old)- and sex-matched WT (n = 18) and Tnfrsf1-dKO (n = 18) mice, and mean (SD) cellularities were determined. (b and c) In two separate experiments, age (8–12 wk old)- and sex-matched adult WT C57BL/6 and Tnfrsf1-dKO mice, five of each genotype, were analyzed individually for frequencies of LSKFLT3− cells by FACS. (b) Representative FACS analysis for each of the genotypes. Displayed percentages of population frequencies of total BM cells are mean values for five mice in each group, each mouse analyzed individually. (c) BM cell numbers and absolute numbers of LSKFLT3− cells were determined for each mouse individually (see Materials and methods). Mean (SD) values of five mice are shown. (d) In two separate experiments with 14 recipients in each, 20,000 unfractionated BM cells from either WT (CD45.1+/2+) or Tnfrsf1-dKO (CD45.1−/2+) mice were transplanted, together with 200,000 competitor BM cells (CD45.1+/2−), into lethally irradiated congenic WT recipients (CD45.1+/2−). 16 wk after transplantation, recipients were analyzed individually for multilineage reconstitution. The percentage of PB donor myeloid reconstitution in positively reconstituted mice is indicated. Horizontal lines represent median reconstitution levels. The frequencies of reconstituted mice (as defined in Materials and methods) out of total transplanted mice are indicated. The indicated p-value is for difference in mean donor reconstitution levels of mice transplanted with Tnfrsf1-dKO and WT BM cells. (e and f) CD45.1+ BM cells from four WT mice and CD45.2+ BM cells from four Tnfrsf1-dKO mice were individually isolated, and subsequently cells from each of the WT replicates were mixed with cells from one of the Tnfrsf1-dKO mice. Cells from each genotype were mixed at equal numbers, and each cell mixture was analyzed individually. Cell mixtures were analyzed by FACS for G0 (DAPIlow/Ki67−), G1 (DAPIlow/Ki67+), and S/G2/M (DAPIhigh/Ki67+) cell cycle stages within the LSKFLT3− HSC compartment. Percentages represent mean (SD) values of all mice.$

HSC numbers are normal in steady-state adult BM of Tnfrsf1-dKO mice. (a) In six separate experiments with three mice in each experiment, BM cells from two tibiae and two femurs per mouse were individually isolated from age (8–12 wk old)- and sex-matched WT (n = 18) and Tnfrsf1-dKO (n = 18) mice, and mean (SD) cellularities were determined. (b and c) In two separate experiments, age (8–12 wk old)- and sex-matched adult WT C57BL/6 and Tnfrsf1-dKO mice, five of each genotype, were analyzed individually for frequencies of LSKFLT3⁻ cells by FACS. (b) Representative FACS analysis for each of the genotypes. Displayed percentages of population frequencies of total BM cells are mean values for five mice in each group, each mouse analyzed individually. (c) BM cell numbers and absolute numbers of LSKFLT3⁻ cells were determined for each mouse individually (see Materials and methods). Mean (SD) values of five mice are shown. (d) In two separate experiments with 14 recipients in each, 20,000 unfractionated BM cells from either WT (CD45.1⁺/2⁺) or Tnfrsf1-dKO (CD45.1⁻/2⁺) mice were transplanted, together with 200,000 competitor BM cells (CD45.1⁺/2⁻), into lethally irradiated congenic WT recipients (CD45.1⁺/2⁻). 16 wk after transplantation, recipients were analyzed individually for multilineage reconstitution. The percentage of PB donor myeloid reconstitution in positively reconstituted mice is indicated. Horizontal lines represent median reconstitution levels. The frequencies of reconstituted mice (as defined in Materials and methods) out of total transplanted mice are indicated. The indicated p-value is for difference in mean donor reconstitution levels of mice transplanted with Tnfrsf1-dKO and WT BM cells. (e and f) CD45.1⁺ BM cells from four WT mice and CD45.2⁺ BM cells from four Tnfrsf1-dKO mice were individually isolated, and subsequently cells from each of the WT replicates were mixed with cells from one of the Tnfrsf1-dKO mice. Cells from each genotype were mixed at equal numbers, and each cell mixture was analyzed individually. Cell mixtures were analyzed by FACS for G0 (DAPI^low/Ki67⁻), G1 (DAPI^low/Ki67⁺), and S/G2/M (DAPI^high/Ki67⁺) cell cycle stages within the LSKFLT3⁻ HSC compartment. Percentages represent mean (SD) values of all mice.

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