CXCL12 stimulates and AMD3100 inhibits osteoclast differentiation. Splenocytes of three collagen type II/complete Freund's adjuvant-immunized mice were isolated and pooled. (a,b) Splenocytes were cultured for 6 days in the cups of a chamberslide, in the presence of the indicated stimuli (macrophage colony-stimulating factor (M-CSF), 20 ng/ml; receptor activator of NF-κB ligand (RANKL), 100 ng/ml; CXCL12, 0.1 μg/ml in (a), 0.5 μg/ml in (b); AMD3100, 25 μg/ml). After stimulation, cells were fixed and stained for the presence of tartrate-resistant acid phosphatase (TRAP). TRAP+ multinucleated (three or more nuclei) cells were counted within each cup. Bars represent averages ± standard error of the mean for four cultures. The asterisk represents p < 0.05 compared with the hatched bar (Mann-Whitney U-test). Representative pictures for TRAP-stained cultures stimulated with (c) M-CSF and RANKL alone and with added (d) CXCL12 (0.5 μg/ml) or (e) CXCL12 and AMD3100.