Figure 5
BMMs cultured in Lm-332-coated plates retain macrophage characteristics in the presence of RANKL. (a) Nonspecific esterase activity was determined using the α-Naphthyl Acetate Esterase Kit (Sigma) according to the manufacturer’s instructions. BMMs exhibit activity of the macrophage-specific enzyme α-NSE, which is detectable by characteristic black cytoplasmic staining (left); bars, 50 μm. The proportion of NSE-positive cells to total cells at day 4 was quantified (right); bars, ±s.d. **P<0.01 by ANOVA with Tukey's test. (b) BMMs were cultured in the presence of M-CSF (20 ng/ml) and RANKL (50 ng/ml) in Lm-332-coated (2–8 μg/ml) or non-coated plates for 1, 2, or 4 days. mRNA expression of macrophage markers (Lyz and Emr1) was analyzed by quantitative real-time PCR. For each target gene, the expression was normalized to B2m, and the expression level is shown relative to the control (designated as 1) for comparison; bars, ±s.d. **P<0.01 by ANOVA with Tukey's test.
