Resorption Pit Assay: An In Vitro Technique to Quantify Calcium Resorption Activity of Mature Osteoclasts using Calcium Phosphate-Coated Culture Plates

Osteoclasts, multinucleated macrophages, facilitate bone resorption - break down of bone tissue and the release of bone minerals.

To quantify osteoclast resorption in vitro, culture human peripheral blood mononuclear cells in macrophage colony-stimulating factor, M-CSF, containing media for differentiation into osteoclast progenitor cells and attachment. Harvest the osteoclast progenitors and centrifuge.

Pipette the resuspended cell suspension in media, supplemented with M-CSF and receptor activator of nuclear factor kappa-Β ligand, RANKL, into multi-well plate wells coated with calcium phosphate, mimicking inorganic bone component. M-CSF and RANKL cause osteoclast progenitor differentiation and fusion into osteoclasts, activating resorption.

Activated osteoclasts attach to the calcium phosphate coating, reorganizing their cytoskeleton to form an actin-rich sealing zone, and develop ruffled borders, which secrete protons and acid hydrolases. The resulting acidic environment dissolves the calcium phosphate coating, releasing calcium and phosphate ions and forming a resorption pit.

Post-incubation, wash the wells, removing dissolved ions. Fix and permeabilize the osteoclasts for subs