In vitro osteoclastogenesis was induced by culturing BMMs in the osteoclastogenic medium (α-MEM containing 30 ng/ml M-CSF and 100 ng/ml RANKL) for 4 days. Fully mature osteoclasts were formed between day 4 and 5. Mature osteoclasts were visualized by TRAP staining and TRAP-positive cells were counted using an Olympus 23 light microscope (Olympus, Tokyo, Japan). TRAP-positive multinucleated (≥4 nuclei) cells were counted as osteoclasts.
23 light microscope
Manufactured by Olympus
Sourced in Japan
The Olympus 23 light microscope is a high-quality optical instrument designed for various laboratory applications. It features an LED illumination system and delivers clear, detailed images for precise observation and analysis. The microscope is equipped with multiple objectives and eyepieces to accommodate a range of magnification requirements.
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3 protocols using 23 light microscope
1
Osteoclastogenesis from Bone Marrow Macrophages
2
Osteoclast Differentiation Assay
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BMMs were seeded at 2 × 104 cells per well and cultured under differentiation conditions. After 4 days, the cells were fixed with 3.7% formaldehyde for 15 min and permeabilized with 0.1% Triton X-100 for 1 min. The cells were treated with tartrate-resistant acid phosphatase (TRAP) staining solution (Sigma) following the manufacturer’s protocol. After 15 min, the TRAP-positive cells were counted under an Olympus 23 light microscope (Olympus, Tokyo, Japan).
3
TRAP Activity Quantification of Osteoclast Differentiation
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BMMs were plated in 96 well plates at the density of 1×104 cells per well. Cells were incubated with M-CSF and RANKL in the absence or presence of CPB at various concentrations. After 4 days of incubation, cells were fixed with 3.7% formaldehyde and permeabilized with 0.1% Triton X-100. Cells were stained for TRAP activity using Leukocyte Acid Phosphatase Kit (Sigma, Cat. No. 387A-1KT) following the manufacturer's protocol. TRAP-positive cells were quantified using the Olympus23 light microscope (Tokyo, Japan).
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