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 subsequent staining. Stain the actin filaments with a suitable dye. Counterstain the nuclei with a DNA-binding dye. Stain the calcium in the well coating with a calcium-binding dye.

Under a fluorescence microscope, functional osteoclasts exhibit multiple fluorescent nuclei and actin rings, crucial for resorptive function. Resorption pits appear as black areas on the fluorescent calcium phosphate coating overlaid by the actin rings.

Using suitable software, quantify the pit area, determining osteoclastic resorption.