Tartrate-Resistant Acid Phosphatase Staining: An In Vitro Technique to Detect TRAP Enzyme-Containing Cultured Osteoclasts

Osteoclasts are large, dome-shaped, multinucleated cells found on the bone surface. These cells contain numerous cytoplasmic granules rich in tartrate-resistant acid phosphatase, or TRAP - a metalloprotein enzyme secreted from the osteoclasts’ ruffled border into the bone tissue. This enzyme digests the extracellular matrix and dissolves the mineral crystals, facilitating bone resorption.

To detect TRAP granules as cytochemical markers of osteoclasts, take a culture dish containing adherent osteoclasts. Add TRAP staining solution to the cells. This solution comprises the substrate - a phosphate-containing naphthol-derivative and tartaric acid in a buffer supplemented with a diazonium salt.

Add acetic acid to this solution to maintain the acidic pH necessary for TRAP staining. Incubate the cells in the dark at physiological temperature for optimum TRAP enzyme activity.

The staining solution enters the osteoclasts, where the tartaric acid inhibits the hydrolyzing activity of tartrate-sensitive acid phosphatases in other cells.

At the same time, TRAP hydrolyzes the added substrate to remove its phosphate group. The hydrolyzed substrate couples with a diazonium salt from the staining solution to form a maroon-colored insoluble dye complex. This complex precipitates as granules at the sites of enzymatic activity.

Image the cells under a microscope. The deposition of maroon dye granules inside the cytoplasm confirms the presence of TRAP-containing osteoclasts in the culture.