Spontaneous death of freshly matured human monocyte-derived DCs. Immediately after maturation, human monocyte-derived DCs were cultured under standard conditions for 4 days and an aliquot of cells was collected every day for cell death determination (0, 1, 2, 3 or 4 days of culture). (A) Flow cytometric analysis of Annexin V-FITC binding and PI staining in cultured DCs at different incubation times (days). The percentage of DCs in each quadrant is indicated. Similar results were obtained from five separate donors. (B) Flow cytometric determination of the percentage of sub-G1 cells obtained by cell cycle evaluation and the percentage of PI permeable cells (PI +) at different culture intervals (days) is shown. Mean ± S.D. from 5 independent experiments are shown. (C) When indicated, cytospin preparations of DCs were stained with May-Grundwald-Giemsa to evaluate general DC morphology (Ao: Apoptotic cell; Nc: Necrotic cell). Micrographs were taken under a transmission electron microscope (original magnification ×630). Two independent experiments gave similar results. (D) Agarose gel electrophoresis of DC DNA before (0) and after (1 or 2 days) culture. Two independent experiments gave similar results.