Fig. 3

Coculture with macrophages enhances osteogenic ability. MC3T3 cells were cultured alone or with polarized macrophages; analyses were performed 2 or 3 weeks after seeding. Week 2 ALP, OC, and OPN gene expression, relative to housekeeping gene 18s and fold change from MC3T3 monocultures in MC3T3 growth medium, were analyzed by qRT-PCR a–c (left panels). Week 2 ALP activity and week 3 OC and OPN protein secretions were quantified by the p-nitrophenyl phosphate method and ELISA, respectively a–c (right panels). Week 3 cultures were stained with Alizarin Red, destained, and quantified by absorbance at 562 nm d. *p <0.05, **p <0.01, ***p <0.001, and ****p <0.0001. ALP alkaline phosphatase, OC osteocalcin, OD optical density, OPN osteopontin