This article discusses the identification of osteoclasts through the detection of tartrate-resistant acid phosphatase (TRAP) granules. The methodology involves specific staining techniques that highlight the presence of these cells in culture.
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.
In this step, aspirate the medium, and wash the sample gently three times with 1X PBS. Next, fix the cells in each well of the 96-well plate with 100 microliters of fix solution for 30 seconds at room temperature.
After fixation, aspirate the fix solution, and wash the cells gently three times with deionized water, pre-warmed to 37 degrees Celsius. Then, aspirate the deionized water, and add 100 microliters of TRAP stain into each well of the 96-well plate.
Place the plate in an incubator at 37 degrees Celsius for 30 minutes, and shield from light. After staining, aspirate the TRAP stain and gently wash three times with deionized water. Subsequently, image the osteoclasts in an inverted microscope at 40X magnification.
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