Evidence that apoptotic cells express podoplanin. (A) Podoplanin expression on the indicated cell lines was assessed by flow cytometry. Black filled histogram: unstained cells, grey line: cells stained with isotype control antibody, grey filled histogram: cells stained with 18H5. Similar results were obtained in two independent experiments. (B) Apoptotic and necrotic CEM×174 express podoplanin. Cultured CEM×174 cells were serum-starved and podoplanin expression on viable and apoptotic cells, as determined by forward and sideward scatter, analyzed by flow cytometry (left panel, the histograms were obtained by gating on dead or live cells, as indicated by the arrows in the scatter plot). Alternatively, the cells were co-stained with podoplanin-specific antibody and the apoptosis marker annexin V or the necrosis marker 7-AAD, and staining analyzed by flow cytometry including both, live and dead cells. Black filled histogram: unstained cells, grey line: cells stained with isotype control antibody, grey filled histogram: cells stained with 18H5. (C) The podoplanin-specific antibody 18H5 was pre-incubated with podoplanin-Fc fusion protein or Fc-control protein before addition to apoptotic cells, and antibody binding was subsequently analyzed by flow cytometry. The results of a representative experiment are shown. Similar results were obtained in three separate experiments. (D) Serum-starved CEM×174 cells were incubated with 1 μM staurosporine for the indicated times and then stained with anti-podoplanin antibody 18H5 and annexin V. Subsequently, podoplanin expression (black bars) and annexin V binding (white bars) were determined by flow cytometry. The results represent the average of two independent experiments and are shown relative to the podoplanin expression and annexin V binding at 0 hrs. The error bars indicate SEM. GMCF: geometric mean channel fluorescence, SEM: standard error of the mean.