Effect of TRAIL on differentiation of adherent PBMCs into functional osteoclasts. Adherent PBMCs were left untreated (without cytokines) or cultured in the presence of TRAIL and with RANKL plus M-CSF with or without TRAIL. After 10 days cells were analyzed for osteoclastic differentiation. (A) Representative fields of the cultures, treated as indicated, after TRAP staining (magnification, × 10; numerical aperture of the objective lens [NA], 0.25). Similar results were observed in 7 independent experiments performed in duplicate. (B) Magnification of a representative TRAP-positive multinucleated cell (i), and a representative field of RANKL plus M-CSF-treated cultures observed by light microscopy (ii) and by fluorescence microscopy after DAPI staining (iii). ^*Polynucleated cells characteristic of RANKL plus M-CSF cultures. Original magnification and NA: i, × 40 0.75 NA; ii-iii, × 20, 0.25 NA. In panel C, adherent PBMCs were plated on an artificial bone matrix slide and were cultured with RANKL plus M-CSF, in the absence or presence of TRAIL (10 ng/mL), as indicated. After 12 days, the slides were fixed and stained, and resorption was determined by examining pit formation under a light microscope (magnification, × 20; NA, 0.40). Representative fields are shown. (D-E) Cultures were treated as indicated and the number of TRAP-positive multinucleated cells containing 3 or more nuclei was scored. Data represent the means ± SD of 3 to 7 different experiments (^*P < .05, compared with RANKL plus M-CSF).