Osteoblast, osteoclast and macrophage differentiation.
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Bone marrow derived monocytes were seeded in 24-well culture plates and induced to differentiate into macrophages or osteoclasts via the addition of M-CSF or M-CSF + RANKL respectively.
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Osteoclastogenesis assays
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Osteoclastogenesis assays were performed as in 5 cells were plated in each well of a 24-well dish with 60 ng/mL RANKL for 3 days.
- After 3 days, 2×103 MDA-231, shControl, or shEGFR-MDA-231 cells were plated with the bone marrow cells, with 60 ng/mL RANKL and 10 ng/mL MCSF and grown for 3 days before TRAP staining.
- For TRAP staining, cells were washed with 1×PBS, fixed with ice cold methanol for 10 minutes, and stained in fresh TRAP solution for 15 minutes at 37°C.
- TRAP solution was replaced with 1×PBS for cell counting under the microscope.
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Osteoclastogenesis assays
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Osteoclastogenesis assays were performed as in 5 cells were plated in each well of a 24-well dish with 60 ng/mL RANKL for 3 days.
- After 3 days, 2×103 MDA-231, shControl, or shEGFR-MDA-231 cells were plated with the bone marrow cells, with 60 ng/mL RANKL and 10 ng/mL MCSF and grown for 3 days before TRAP staining.
- For TRAP staining, cells were washed with 1×PBS, fixed with ice cold methanol for 10 minutes, and stained in fresh TRAP solution for 15 minutes at 37°C.
- TRAP solution was replaced with 1×PBS for cell counting under the microscope.
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PLCγ2 is required for in vitro osteoclast development.
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(a) Representative TRAP-stained images of wild-type and PLCγ2−/− bone marrow cells cultured in the presence of the indicated concentrations of recombinant murine M-CSF and RANKL for 4 days.
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Expression of pro-resorptive cytokines in gouty tophus tissues.
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(c) Immunofluorescent stains for CD3 (green) and RANKL (red) in tophaceous gout tissues.
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Polarized membrane extensions mediate osteoclast fusion.
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(D, top) Immunoblot analysis of lysates of RAW264.7 macrophages stimulated with RANKL for the indicated times with antibodies to Ser473-phosphorylated (p) or total forms of Akt.
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Osteoclast differentiation assay
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Osteoclast differentiation assay was performed as we described.+ T cells or Treg cells (1 × 106/ml) were pre-activated with 2.5 μg/ml anti-CD3 and 1.25 μg/ml anti-CD28 monoclonal mouse antibodies for 12 h as we described,,
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Inositol hexakisphosphate (IP6) directly inhibits osteoclast formation induced by RANKL.
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RAW 264.7 cells cultured for 5 days with no stimulation with RANKL (left).
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Inositol hexakisphosphate (IP6) directly inhibits osteoclast formation induced by RANKL.
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RAW 264.7 cells cultured for 5 days with no stimulation with RANKL (left).
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Cofilin is activated when podosome belts are assembled.
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BMMs were cultured for 3 days in the absence or in the presence (d1 to d3) of RANKL.
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