Fig. 3
From: A novel method to efficiently differentiate human osteoclasts from blood-derived monocytes
Cell density and concentration of M-CSF and RANKL are crucial for osteoclast differentiation. A + B Immunofluorescence: Isolated monocytes were seeded at different cell densities (indicated as cells/cm2) and stimulated with either a low (5 ng/ml M-CSF, 10 ng/ml RANKL) or high (25 ng/ml M-CSF, 50 ng/ml RANKL) concentration of cytokines. Mature osteoclasts were assessed by staining for F-Actin (green) and DAPI (blue) (A). White arrows point at cells with ≥ 3 nuclei (scale bar: 50 µm). The proportion of multinucleated cells was counted and compared between the different conditions (Boxplots depict the mean (line) and the 25–75 percentiles (box) of 3 data points) (B). C Immunofluorescence: Isolated monocytes were differentiated to osteoclasts according to the standard (plate) or novel (bag) protocol. Macrophage differentiation in plates was performed with cells either cultivated in their normal growth medium or in medium additionally supplemented with 2% AB serum. White arrows point at cells with ≥ 3 nuclei. D Confocal microscopy: Monocytes (2.0 × 10.5) from cell culture plates or Teflon bags were seeded on glass coverslips, differentiated to osteoclasts in the presence of high concentrations of M-CSF and RANKL and stained for F-Actin (green) and DAPI (blue) (scale bar: 10 µm)