CBNPs cause acute necrosis of alveolar macrophages in vivo. C57BL/6 mice were exposed to a single instillation of saline containing 0.2 mg of CBNPs. (A) The detection of LDH release in BALF 30 min after instillation. (B) The representative images of PI-positive necrotic cells in mouse lungs. Scale bar = 50 µm. (C) The detection of the necrotic cells induced by CBNPs in BALF by flow cytometry with Annexin V and PI staining. Percentages of necrotic cells in PI-positive and Annexin V-negative region are shown. (D-E) Four hours after instillation, the detection of necrotic macrophages in BALF induced by CBNPs and the representative experiments by flow cytometry with PI and F4/80 staining are presented. (F-H) After 24 h of instillation, alveolar macrophages were obtained from the BALF of the exposed mice and the cells were stained by H&E (F), trypan blue (G) and PI (H). Scale bar = 50 µm. Data are mean ± SEM; n = 3 or 4 per group, *P < 0.05, ***P < 0.001 compared with the control group by Student's t-test.