Ceramide is elevated in CD44+ cells, which is suppressed by DC. (a) 4T1 cells were cultivated in serum-free medium and immunocytochemistry was performed for ceramide (blue) and CD44 (red). Apoptotic cells were identified by TUNEL staining (green). Note that CD44+ cells contained high levels of ceramide, which induced apoptosis in the periphery of CD44+ cell clusters (left panel). Ceramide elevation and apoptosis was prevented by DC (right panel). Scale bar = 100 μm. (b) Immunocytochemistry as in panel a (without DC), showing three optical planes as obtained from confocal laser scanning immunofluorescence microsocopy (Z-scan). Note that CD44+/ceramide+/TUNEL+ cells are located at the periphery of CD44+ cell clusters. Scale bar = 20 μm. (c) Immunocytochemistry as in panel a, showing Hoechst staining of nuclei. Note that the majority of the CD44+/ceramide+ cells were apoptotic (TUNEL+), unless cells were incubated with DC (panels on the right). Scale bar = 10 μm. (d) Confocal scanning as described in panel b, but reconstruction of the fluorescent signal distribution in a Z-scan (z-axis is shown). Note that the peripheral distribution of ceramide+/TUNEL+ cells is consistent with the results shown in panel b. Scale bar = 10 μm. DC, sodium deoxycholate; TUNEL, terminal dUTP nick-end labeling.