CD8α+ dendritic cells (DC) selectively capture apoptotic cells and cross-present cell-associated antigen on major histocompatibility complex (MHC) class I in vitro. (a) Sorted CD4+ (open bars) or CD8α+ (filled bars) DC subsets (C57BL/6) were co-cultured with OT-I T cells in the presence of irradiated, ovalbumin (OVA)-loaded allogeneic (BALB/c) splenocytes. Results represent the mean [3H]thymidine uptake of triplicate wells. All error bars are shown and represent 1 SD from the mean. c.p.m., counts /min. (b)–(d) Flow cytometry analysis of 3-hr cultures containing prestained, CD11c-enriched cells and PKH26-labelled RAW264.7 cells, as indicated. (b) Ultraviolet (UV)-treated RAW cells were added to DC cultures in the presence or absence of sodium azide (top) or were first cultured and then sorted into annexin V+ or annexin V− (AxV+, AxV−, respectively) fractions and fixed in paraformaldehyde (PFA), as described in the Materials and methods (bottom). Dot-plots represent CD8α expression versus PKH26 fluorescence for DC gated on the basis of high CD11c expression. Numbers show the percentage of cells within each quadrant gate. (c) Histograms showing PKH26 profile for CD8α+ (top), CD4+ (middle) and double negative (DN, bottom) DC subsets from cultures with (thick line) or without (thin line) PKH26-labelled UV-treated RAW cells. (d) Frequencies of PKH26+ DC subsets after incubation with increasing doses of UV-treated, PKH26-labelled RAW cells. Data are representative of (a) six experiments, (b) three experiments, or (c) and (d) two experiments.