Analysis of apoptotic neutrophils in the different lung compartments, platelet–leukocyte aggregates in the blood, and the BAL by flow cytometry. (A) Exemplary flow cytometry plots and gating strategy of lung homogenates. (B and C) Exemplary flow cytometry plots and gating strategy of (B) platelet–neutrophil aggregates and (C) platelet–T reg cell aggregates. (D) Apoptosis rate in CD41^neg (without bound platelet) and CD41^pos (with bound platelet) neutrophil subpopulation in the BAL obtained from WT mice after induction of bacterial pneumonia (data pooled from two independent experiments with four to five mice in each group; Mann-Whitney test was used for statistical analysis, *, P < 0.05). (E) Platelets in the BAL fluid were analyzed by flow cytometry after induction of pneumonia (exemplary flow cytometry plots). Isolated WT platelets were stimulated with different ADP concentrations and platelet P-selectin surface expression was analyzed by flow cytometry. (F and G) Quantitative comparison of mean fluorescence intensity (MFI; F) and gating strategy and histogram of CD62-PE binding (G; data pooled from three independent experiments with six samples in each group; Mann-Whitney test was used for statistical analysis, *, P < 0.05). (H) BAL was obtained 3 d after induction of bacterial pneumonia in WT mice and platelets CD62-PE binding was analyzed and compared with resting and thrombin-stimulated WT platelets isolated from peripheral blood by flow cytometry (exemplary FACS plot from four experiments). FMO, fluorescence minus one sample of corresponding BAL sample; FSC, forward scatter; SSC, side scatter.