Fig. 4.

TIM-4^−/− peritoneal macrophages cannot efficiently engulf apoptotic cells in vitro or in vivo. (A) Peritoneal cells were cultured with CMFDA-labeled AB for various time points and analyzed by flow cytometry. Gates included CD11b⁺F4/80⁺ cells (Top) and CD11b⁺CD19⁺ cells (Middle). The average frequency of F4/80⁺CD11b⁺ that were CMFDA-AB⁺ after 30, 60, or 90 min is shown (Bottom). Data represent six independent experiments. (B) Peritoneal cells were cultured with CMFDA-labeled AB (green) for 3 h. Cells were stained with CD11b-PE (red) and analyzed by immunofluorescence microscopy. Arrows indicate nonengulfed AB associated with macrophage cell surface. Frequencies of CD11b⁺ cells that engulfed AB in two independent experiments are summarized (Right). (C) Images representing a 90-min time-lapse video of peritoneal cells cultured with CMFDA-labeled AB. Arrows indicate peritoneal cells associating with AB. (D) Representative images from a Z-stack analysis performed on peritoneal cells that were treated as in B. Images in C and D are representative of two independent experiments. (E) Mice were injected i.p. with CMFDA-labeled AB and killed 30 min later. Peritoneal cells were harvested and analyzed by flow cytometry (Top). Gate includes CD11b⁺F4/80⁺ cells. Data from three independent experiments are summarized (Bottom; *P < 0.05).