Lower frequency and higher apoptosis of splenic early erythroblast subsets in the basal state. (A) The ProE and Ery.A subsets are a lower proportion of Ter119⁺ cells in spleen than in bone marrow. Data are the mean ± SEM from Balb/C mice (n = 7). ^*Student t test (2-tailed, unequal variance), P < .01. (B) Higher annexin V binding of splenic Ery.A compared with equivalent bone marrow cells. Viable cells (impermeable to 7-AAD) from either bone marrow (top panels) or spleen (bottom panels) were analyzed for Ter119/CD71 expression. Ter119^high cells were gated and further analyzed with respect to FSC. FSC^highCD71^high cells (Ery.A) were examined for annexin V binding (right panel). Background fluorescence in the annexin V channel was determined by comparison with Ery.A cells stained for all colors except for annexin V (“fluorescence minus one” or FMO-annexin V, gray curve). The following fluorescent conjugates were used: CD71-FITC, Ter119-APC, 7-AAD, annexin V-Alexa Fluor 350. Controls included unstained cells, single-color controls, and FMO controls. (C) Higher annexin V binding in splenic erythroblasts compared with equivalent bone marrow erythroblasts. Staining strategy and data analysis as in panel B, for each of the indicated erythroblast subsets. Mean ± SEM, Balb/C mice (n = 4); ^*P < .003; ^**P < .001. (D) Higher proportion of cells positive for activated caspase 3 in splenic than in bone marrow Ery.A. The proportion (%) of cells staining positive for caspase 3 in each tissue is indicated. Representative experiment; tissue from 2 Balb/C mice.