Fig 5. Stimulatory effect of acidic extracellular pH on osteoclast differentiation.

Osteoclast precursors were cultured under a CO₂-free condition in HEPES-buffered medium (pH 7.0 or 7.5) in the presence of M-CSF (30 ng/ml) and RANKL (100 ng/ml) for 4 days (A) or for the indicated times (B). (A) Measurement of osteoclast formation. Cells were stained with TRAP and the number of TRAP⁺ MNCs with more than three nuclei was counted under a light microscope. Scale bar, 100 μm. (B) Analysis of osteoclastogenic gene expression. mRNA levels were analyzed using quantitative real-time PCR.