Fas and FasL are coexpressed by early erythroblasts and mediate early erythroblast apoptosis in vivo. (A-B) Expression of Fas (A) or FasL (B) in spleen and bone marrow cells. Both Fas and FasL are expressed at higher frequencies in ProE and Ery.A subsets. Both are also expressed at higher frequencies in spleen than in bone marrow. Freshly isolated spleen and bone marrow were labeled with antibodies against Ter119, CD71, and either Fas (Jo2) or FasL (MFL3). Staining strategy and analysis were similar to those described in Figure 3. Data from C57BL6/129 mice, n = 5, mean ± SEM. ^*P < .004; ^**P < .001. (C) Fas and FasL are coexpressed by Ery.A in spleen (top left panel) and bone marrow (top right panel). Ter119/CD71/FSC analysis was used to gate Ery.A, as illustrated in Figure 1. Bottom panels show FMO controls (Figure 3B), which were used to set background fluorescence in Ery.A. These lack either Fas or FasL primary antibodies, as indicated, but contain secondary and all other primary antibodies. Representative of 3 experiments. Two different combinations of fluorescent conjugates gave similar results. (D-E) Single-cell analysis of Fas expression and annexin V binding in spleen and bone marrow Ery.A. Freshly isolated bone marrow and spleen cells were simultaneously labeled with annexin V and with antibodies to Ter119, CD71, and Fas. Fas-positive Ery.A are several-fold more likely to bind annexin V than Fas-negative cells (D). In panel E, Ery.A were subdivided into 8 subsets of increasing Fas expression, measured as geometric mean fluorescence per cells. Fas expression is correlated with annexin V binding, in both spleen and bone marrow. Data are from Balb/C mice (n = 2).