Enhanced apoptosis in Mcl-1^−/− neutrophils. (A) Detection of apoptosis in granulocytes and macrophages of Mcl-1^−/− mice. Apoptosis was detected by double staining of 7-AAD and annexin V. Mac-1⁺Gr-1⁺ and Mac-1⁺Gr-1⁻ cells from BM, blood, spleen, and peritoneal cavity day 1 and day 4 after thioglycollate injection (PEC d1, d4) were gated for 7-AAD and annexin V staining. Numbers indicate percents in each region. The results are representative of 6 independent experiments from 12 mice. (B) Apoptosis rates of Mcl-1–deficient BM neutrophils with or without growth factor stimulation. BM cells from Mcl-1–deficient (KO) and control mice were stimulated with G-CSF or GM-CSF for different periods of time in triplicates. Mac-1⁺Gr-1⁺ cells were examined for apoptosis by flow cytometry. The graph shows the mean and standard deviation of the percentage of apoptotic and dead cells as defined by annexin V⁺. The results are representative of 3 independent experiments. (C) Mcl-1 expression in purified BM neutrophils treated with 10 ng/mL GM-CSF for 24 hours. Relative protein expression was normalized to Erk-2 expression levels. (D) Western blot analysis of Bcl-2 and Bcl-x_L expression in Mcl-1^−/− macrophages. BM macrophages were lysed and blotted with anti–Bcl-2 and Bcl-x_L antibodies as shown in Figure 1D.