Induction of apoptosis by FcαRI targeting in human monocytes and FcαRI RBL-2H3 transfectants. (A-B) Analysis of annexin V/PI double-positive populations in human blood monocytes (A) and RBL-2H3 cells expressing WT FcαRI (B) after 24 hours of treatment with 10 μg/mL anti-FcαRI (A77) Fab or control (320) Fab. Cells were stained with annexin V and PI and analyzed by flow cytometry as described in “Materials and methods.” The mean ± SD percentage of annexin V/PI double-positive cells treated with PBS, 320, or A77 Fab in 5 experiments is shown. *P < .05. (C) Kinetic and dose-response analysis of annexin V/PI double-positive populations of WT FcαRI RBL-2H3 transfectants after treatment with 10 μg/mL anti-FcαRI (A77) Fab or control (320) Fab for 1 to 24 hours or for 24 hours with the indicated concentrations of A77 or 320 Fab. The percentage of annexin V/PI double-positive populations was determined as described in panel A. (D) Analysis of hypoploid nuclei in blood monocytes and WT FcαRI RBL-2H3 transfectants after treatment with 10 μg/mL anti-FcαRI (A77) or control (320) Fab for 24 hours. Cells were stained with PI and analyzed for the appearance of hypoploid nuclei as described in “Materials and methods.” Numbers indicate the percentage of cells with hypoploid nuclei (a sign of apoptosis). (E) Analysis of condensed apoptotic nuclei in WT FcαRI RBL-2H3 transfectants after 24 hours of treatment with 10 μg/mL anti-FcαRI (A77) Fab or control (320) Fab. Following treatment, cells were stained with DAPI as described in “Materials and methods,” before analysis by fluorescence microscopy (top panels; 40×/1.3 NA objective lens; insets represent a zoom of one of the nuclei.). Apoptotic cells have condensed nuclei and crescent shape (inset). Treated cells were also examined by light microscopy (lower panels, 40×/1.3 NA objective lens). Apoptotic cells contained large vacuoles (inset).