Stimulation with CCL19 and CCL21 reduces the percentage of apoptotic DCs. (A) Cells were washed and then incubated for 6 hours in 0.1% BSA in RPMI in the absence (control) or in the presence of 200 ng/mL CCL19, CCL21, or 10% fetal calf serum (FCS). (i) DCs were analyzed for annexin V (FL1-H) and 7-amino actinomycin (7-AAD) (FL3-H) by flow cytometry. To exclude necrotic cells (7-AAD–positive), only annexin V–positive/7-AAD–negative cells were considered apoptotic. (ii) Quantification of the percentage of annexin V–positive/7-AAD–negative cells. Results represent the mean ± SEM (n = 8). (B) Apoptotic cells were also stained with DePsipher to detect loss of mitochondria potential and then analyzed by flow cytometry (i). In this experiment we used as positive control cells treated with carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), a protonophore that dissipates the H⁺ gradient across the inner membrane of mitochondria and induces apoptosis. Figure viewed with the Nikon Diaphot microscope. (ii) DePsipher immunofluorescence staining. Healthy cells that were stained with DePsipher give an intense red labeling (observed as a bright labeling on this black-and-white figure) under the fluorescent microscope. Figure viewed with the Nikon Diaphot microscope. (C) Scanning electron microcopy of a representative apoptotic cell presenting numerous apoptotic blebs (control) and a CCL19-treated and CCL21-treated cell. This figure was viewed with the Stereoscan 250 microscope. The number of cells presenting blebs was reduced by half in the chemokine-treated cells (not shown). (D) Photographs taken from cells stained with Hoechst 33342. Arrowheads point to condensed or fragmented nuclei.