Fig 4.

M. bovis BCG triggers apoptosis of infected macrophages. (A) Macrophages were infected with M. bovis BCG for indicated times. After infection, cells were washed with PBS and stained with annexin V-FITC for 30 min followed by fixation in 3.7% paraformaldehyde. The cells were analyzed by confocal microscopy. The representative images are shown. Data are representative of 4 separate experiments. (B) Quantification of mean fluorescence intensities of panel A confocal images (data are means ± SE, n = 4). (C and D) After infection, macrophages were fixed with 70% ethanol to leak out apoptotic DNA fragments and total DNA content was visualized by confocal microscopy after staining cells with PI. Shown are a representative of 3 independent experiments (C) and quantification of the same (D). (E) Macrophages were infected with M. bovis BCG for indicated times, fixed in 70% ethanol, stained with PI, and analyzed by flow cytometry. A representative result of 3 experiments is shown. The sub-G₁ subpopulation is indicated by M1, and the percentage of total cells undergoing apoptosis is indicated. Med, medium; BF, bright field; PI, propidium iodide.