IL-4 inhibits osteoclast differentiation by acting directly on progenitor cells. Cells were stained for TRAP, and osteoclasts were identified as multinucleated TRAP-positive (red-stained) cells. (A) CD14⁺ human monocytes were cultured with rhM-CSF (20 ng/mL) and RANKL (150 ng/mL) ± rhIL-4 (10 ng/mL) for 5 days. (B) RAW264.7 cells were cultured with rmM-CSF (20 ng/mL) and RANKL (150 ng/mL) ± rmIL-4 (10 ng/mL) for 5 days. At the end of all culture periods, the plates were stained for TRAP. (C) BMMs were isolated from wild-type or (D) STAT6^-/- mice and were cultured for 3 days in the presence of rmM-CSF (20 ng/mL) to obtain a homogeneous population of M-CSF–dependent adherent macrophages. They were then differentiated to osteoclasts by culturing for 7 days in the presence of rmM-CSF (20 ng/mL), RANKL (150 ng/mL), and rmIL-4 (10 ng/mL). Panel C inset shows a magnified image of an MNG (original magnification, × 20). Original magnification, × 10 for panels A-D.