Apoptotic cells stably bind to PTX3 and influence its production by immature DCs. DCs challenged with LPS (10 ng/mL), early or late apoptotic cells, or a combination of these stimuli release PTX3 (y-axis; nM) in the microenvironment (A). Results representative of three independent experiments are depicted as mean ± SD of duplicate samples. ^*Statistically significant value; P < .05. Exogenous biotinylated PTX3 binds to late (C) but not to early (B) apoptotic Hela cells as evaluated by flow cytometry after addition of FITC-labeled streptavidin (filled profiles). The empty profiles indicate the fluorescence detected in the presence of the second-step reagent only. PTX3 binds to membrane domains of late apoptotic cells (green) and fails to recognize nuclear domains (DAPI, blue), as assessed by confocal scanning laser microscopy (D). Exogenous biotinylated PTX3 binds to viable immature DCs, as evaluated by flow cytometry after addition of FITC-labeled streptavidin (filled profile). The empty profile refers to the fluorescence in the presence of the second-step reagent only (E). PTX3 remains associated to apoptotic cells at different time points (x-axis, hours). Results are expressed as mean fluorescence intensity (MFI; y-axis) assessed by flow cytometry as described above on apoptotic (♦) or living (⋄) HeLa cells (F).