Uptake of apoptotic cells by DCs, Mø1s, and Mø2s. (A) Jurkat T cells were labeled with CFSE and induced into apoptosis by treating cells with etoposide for 18 hours. Apoptotic cells (0.1 × 10⁶ cells) were coincubated with DCs, Mø1s, or Mø2s at a 1:1 ratio, for 0.5 hours and 2 hours at 37°C or 4°C. Prior to flow cytometry analysis, cells were stained with PE-conjugated mAb against CD11b. CFSE and CD11b double-positive populations represent the phagocytes that have bound and/or ingested apoptotic cells. Dot-plots represent the phagocytosis of apoptotic cells at 2 hours. (B) The percentages of uptake and binding (at 37°C) or binding (at 4°C) were calculated as 100% × [(CD11b⁺CFSE⁺)/CD11b⁺]. Data indicate the mean ± SD from 4 independent experiments performed in duplicate. Statistics were performed with 2-way ANOVA. *P < .01; **P < .001. (C) Confocal microscopy images show the uptake of apoptotic cells by Mø1s and Mø2s (see arrows). Red cells represent the CD11b-PE–positive Møs, and green cells are the CFSE-labeled apoptotic cells. Magnification, 400 ×. (D) Based on the confocal images, more than 600 cells of Mø1s and Mø2s were scored. Data are presented as phagocytic index (percentage of phagocytosing Møs × average number of apoptotic cells per Mø). A chi-square test was performed to evaluate the difference in the capacity of apoptotic cell uptake between Mø1s and Mø2s (P < .001). (E) Sheep erythrocytes were opsonized with rabbit anti–sheep red blood cell IgG (E_IgG) and cocultured with Mø1s and Mø2s on Lab-TEK chamber slides at 37°C for 0.5 hours, followed by May-Grünwald/Giemsa staining. Pictures show that E_IgG were ingested by Mø1s and Mø2s (arrows). Magnification, 400 ×. (F) More than 300 single cells of Mø1 or Mø2 were scored by light microscopy, and the phagocytosis of E_IgG was presented as phagocytic index. Data are representative of 3 independent experiments using cells generated from 3 unrelated donors.