Irreversible commitment of BMMs to osteoclasts during the first 48 hours. BMMs from wild-type mice were cultured in the presence of rmM-CSF (20 ng/mL) and RANKL (150 ng/mL) in the presence or absence of rmIL-4 (10 ng/mL) for various time periods, as indicated. (A) BMMs without rmIL-4. (B) rmIL-4 was present only during the first 48 hours. Then rmIL-4 was removed by washing the wells 3 times with α-10 media, and the cultures continued in this media and were complemented with rmM-CSF (20 ng/mL) and RANKL (150 ng/mL) for 5 days. (C) After 2 days of culture in the presence of RANKL and rmM-CSF, rmIL-4 (10 ng/mL) was added to the cultures for 5 days. (D) rmIL-4 was present throughout the culture period. At the end of the culture period, cells were fixed and stained for TRAP. TRAP stain; original magnification, × 10 for all panels.