ATP treatment induces morphologic changes in DCs compatible with apoptosis.

DC suspension was incubated with ATP (5 mmol/L) for 30 minutes at 37°C, washed twice with PBS, and incubated additionally for 6 hours in complete RPMI 1640 medium. DCs were incubated immediately before the analyses with acridine orange (5 μg/mL) and propidium iodide (10 μmol/L) to determine nuclear morphology and cell integrity, respectively. The DC apoptotic phenotype was ascertained by laser scanning confocal microscopy. The right panels represent the images of fluorescence data and the left ones represent the corresponding transmitted images. (A) Control DCs were viable, presenting green-labeled round nuclei. (B) The ATP-treated DC suspension presented cells with features associated with different stages of apoptosis: chromatin condensation (increased nuclei fluorescence), fragmented nuclei and apoptotic bodies (arrows), and secondary necrosis (fragmented nuclei labeled red with propidium iodide). Bar: 10 μm.