Figure 3 Flow cytometry analysis of BM... | American Society of Hematology

Figure 3

$Figure 3. Flow cytometry analysis of BM erythropoiesis and ERFE expression. (A) Identification of clusters of BM erythroid precursors in WT littermates. (Left) Identification of SSC low CD11b− and B220− cells (gate 1). (Middle) Recognition of erythroid precursors (TER119+ cells, gate 2) inside the population identified in gate 1. (Right) Density plot of CD44 vs FSC of cells identified in gate 2 showing naturally occurring clusters of erythroid precursors at progressive maturation stages (fractions I-V). (B) Density plots of TER119+ cells in WT littermates fed the IB or ID diet and in Tmprss6 KO mice on a mixed genetic background showing representative distribution of erythroid precursors. (C) Density plots showing representative distribution of TER119+ cells after treatment with EPO for 15 hours. (D) Quantification of BM erythroid fractions I-V with respect of total BM cells. (E) Quantification of spleen erythroid fractions I-V identified as in (A) on total spleen cells. Quantification was performed on samples from at least 6 mice for every condition. Each erythroid fraction is calculated as a percentage of total BM or spleen cells. Quantitative variations of each erythroid fraction among different mice groups are listed in supplemental Tables 1-4. (F) Tmprss6 KO animals, on a mixed genetic background, were analyzed in comparison with control littermates fed an IB or an ID diet for 3 weeks. Erfe expression was analyzed by qRT-PCR in BM derived cells from 6 to 3 mice per group. mRNA expression was normalized relative to the erythroid marker GypA. Error bars indicate ± SEM. *P < .05; **P < .01; ***P < .001. FSC, forward side scatter; ns, nonsignificant; SSC, side scatter.$

Flow cytometry analysis of BM erythropoiesis and ERFE expression. (A) Identification of clusters of BM erythroid precursors in WT littermates. (Left) Identification of SSC low CD11b⁻ and B220⁻ cells (gate 1). (Middle) Recognition of erythroid precursors (TER119⁺ cells, gate 2) inside the population identified in gate 1. (Right) Density plot of CD44 vs FSC of cells identified in gate 2 showing naturally occurring clusters of erythroid precursors at progressive maturation stages (fractions I-V). (B) Density plots of TER119⁺ cells in WT littermates fed the IB or ID diet and in Tmprss6 KO mice on a mixed genetic background showing representative distribution of erythroid precursors. (C) Density plots showing representative distribution of TER119⁺ cells after treatment with EPO for 15 hours. (D) Quantification of BM erythroid fractions I-V with respect of total BM cells. (E) Quantification of spleen erythroid fractions I-V identified as in (A) on total spleen cells. Quantification was performed on samples from at least 6 mice for every condition. Each erythroid fraction is calculated as a percentage of total BM or spleen cells. Quantitative variations of each erythroid fraction among different mice groups are listed in supplemental Tables 1-4. (F) Tmprss6 KO animals, on a mixed genetic background, were analyzed in comparison with control littermates fed an IB or an ID diet for 3 weeks. Erfe expression was analyzed by qRT-PCR in BM derived cells from 6 to 3 mice per group. mRNA expression was normalized relative to the erythroid marker GypA. Error bars indicate ± SEM. *P < .05; **P < .01; ***P < .001. FSC, forward side scatter; ns, nonsignificant; SSC, side scatter.

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