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

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.