Erythropoiesis in BM and the spleen of SHPS-1 mutant mice. (A) BM cells freshly isolated from the femur of WT or homozygous SHPS-1 mutant (KO) mice at 6 weeks of age were labeled with a PE-conjugated mAb to Gr-1 (thick traces) and an FITC-conjugated mAb to Ter-119 (thick traces). The specific mAb (to Gr-1 or to Ter-119) was replaced with rat IgG to TNP as a negative control (thin traces). The cells were then analyzed by flow cytometry. Mature RBCs (with low forward scatter and low side scatter) were excluded from analysis. The relative numbers of Gr-1-positive or Ter-119-positive cells were expressed as a percentage of all BM cells, as indicated on each histogram. Data are representative of 3 separate experiments. (B) The total BM-cell number (including mature RBCs) per femur was determined with a Burker-Turk counting chamber for WT and SHPS-1 mutant mice (left). The total BM-cell number excluding mature RBCs was calculated according to the density plot of FACS analysis for gating out of mature RBCs. Data similar to those shown in panel A were used to determine the absolute values for Gr-1-positive myeloid cells and Ter-119-positive erythroid cells (middle and right, respectively) by multiplying the percentage of Gr-1-positive myeloid cells or Ter-119-positive erythroid cells by the total BM-cell numbers excluding mature RBCs. Results are means ± SE of values from 3 mice of each genotype. ^*P < .05. (C) Freshly isolated splenic cells from WT or SHPS-1 mutant mice at 6 weeks of age were labeled with a PE-conjugated mAb to CD71, an FITC-conjugated mAb to Ter-119, and 7-AAD. They were then subjected to 3-color flow cytometry, with dead cells (7-AAD positive) and mature RBCs (with low forward scatter and low side scatter) being excluded from the analysis. Without gating out of mature RBCs, the proportion of region III (which represents Ter-119^highCD71^low cells) becomes extremely high (53.42%, without gating out vs 16.14%, with gating out) in the WT animal for instance, suggesting that gating out of mature RBCs is effective and thus essential for the analysis. Results are presented as density plots of all viable splenocytes; axes represent relative fluorescence units for FITC (Ter-119) and PE (CD71). Regions I to III were selected as indicated. The relative numbers of cells in each region were expressed as a percentage of all viable splenocytes, as indicated on each plot. Data are representative of 3 separate experiments. (D) Data similar to those shown in panel C were used to determine the percentages of nonerythroid cells (Ter-119 ^low, region I) as well as the percentages of early erythroblast (Ter-119^highCD71^high, region II) and the percentages of late erythroblast (Ter-119 ^highCD71^low, region III) among total splenocytes for WT and SHPS-1 mutant mice (left). The ratio of Ter-119^high cells (regions II and III) to Ter-119^low cells (region I) and that of early (region II) to late (region III) erythroblasts are shown in the right panel. Results are means ± SE of values from 3 mice of each genotype. ^*P < .01.