Fig 1.
Effects of different concentrations of RANKL alone on osteoclast differentiation in RAW264.7 cells.
RANKL (50 ng/ml and 100 ng/ml) induced differentiation of osteoclasts from RAW264.7 cells for 3 days or 5 days. (A) multinucleated osteoclasts were observed by light microscopy on day 5, original magnification of ×200 (Scale bar = 200 μm). (B) TRAP-positive cells area was analyzed and quantified. The expressions of CTSK (C), c-Fos (D) and NFATc1 (E) were tested by qPCR on day 3. Data are presented as mean ± SD (n = 3). #P<0.05, ##P<0.01, ###P<0.001 vs. 0ng/ml RANKL group; *P<0.05, **P<0.01, ***P<0.001 vs. 50ng/ml RANKL group; ns, no significance.
Fig 2.
Effects of M-CSF on the differentiation of RAW264.7 cells into osteoclasts.
RANKL (50 ng/ml and 100 ng/ml) plus 50 ng/ml M-CSF induced differentiation of osteoclasts from RAW264.7 cells for 3 days or 5 days. (A) multinucleated osteoclasts were observed by light microscopy on day 5, original magnification of ×200 (Scale bar = 200 μm). (B) TRAP-positive cells area was analyzed and quantified. The expressions of CTSK (C), c-Fos (D) and NFATc1 (E) were tested by qPCR on day 3. Data are presented as mean ± SD (n = 3). #P<0.05, ##P<0.01, ###P<0.001 vs. Control group (M-CSF-, RANKL-); *P<0.05, **P<0.01, ***P<0.001 vs. M+R(50ng/ml) group (50ng/ml M-CSF, 50ng/ml RANKL); ns, no significance.
Fig 3.
Effects of different induction conditions on bone resorption pits.
RAW264.7 cells were plated on bovine bone slides with different concentrations of RANKL with or without M-CSF for 7 days. (A) Bone resorption pits were detected by SEM on day 7, original magnification of ×300 (Scale bar = 100 μm), red arrow indicates bone resorption pits. (B) Quantitative analysis of bone resorption pit area. Data are presented as mean ± SD (n = 3). Statistically significant (***P<0.001).
Fig 4.
Effects of different cell densities on differentiation of RAW264.7 cells into osteoclasts.
(A) Multinucleated cells were stained by TRAP staining and observed under light microscopy, original magnification of ×200 (Scale bar = 200 μm). (B) TRAP-positive cells area was analyzed and quantified.