RAW 264.7 cells were seeded in 24-well plates at a density of 2 × 103 cells per well. Negative control lentiviral vector (A549_shNC) and GNAQ short hairpin RNA lentiviral vector (A549_shGNAQ) cells were subsequently seeded in minimum essential medium-alpha modification(α-MEM) (HyClone Laboratories) overlaying the RAW 264.7 cells at a density of 2 × 103 cells per well. Cells were treated with RANKL (50 ng/ml) and M-CSF (20 ng/ml) (R & D Systems, Minneapolis, Minnesota, USA) to stimulate osteoclast differentiation. After five days, the cells were fixed and stained for tartrate-resistant acid phosphatase (TRAP) activity using a TRACP Assay Kit (Takara Bio, Mountain View, California, USA), as per the manufacturer’s instructions. For quantification, osteoclasts were defined as multinucleated (more than three nuclei) TRAP-positive cells.9 (link) Cells were visualized at 200 × magnification using a light microscope. Electronic images of five pre-determined areas per well were obtained, and TRAP-positive cells were counted in each image.
Tracp assay kit
Manufactured by Takara Bio
Sourced in United States, Japan
The TRACP assay kit is a laboratory tool used to measure the activity of tartrate-resistant acid phosphatase (TRACP), an enzyme that can be used as a biomarker for various biological processes. The kit provides a quantitative colorimetric method for determining TRACP levels in biological samples.
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3 protocols using tracp assay kit
1
Osteoclastogenesis Assay in RAW 264.7 Cells
2
Quantitative Osteoclast Differentiation Assay
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Tartrate-resistant acid phosphatase (TRACP) enzyme activity was measured to quantitatively evaluate osteoclast differentiation activity. Cells were treated with RANKL for three and five days, and TRACP activity was measured using the TRACP assay kit (Takara, Kyoto, Japan) according to the manufacturer’s protocol. The TRACP activities of the cultured cells were examined by a colorimetric assay utilizing the conversion of colorless p-nitrophenol phosphate to colored p-nitrophenol. The concentration of p-nitrophenol was measured spectrophotometrically by taking sample absorbance readings at 405 nm (Bio-Rad, Hercules, CA, USA). Replicate measurements were performed with 4 disks for each group.
3
Osteoclastogenesis Induction and Quantification
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The osteoclastogenesis was induced using 70 ng mL−1 of receptor activator of nuclear factor‐κB ligand (RANKL) and 50 ng mL−1 of macrophage colony‐stimulating factor (M‐CSF). To differentiate osteoclast precursor cells into activated osteoclast, the RAW264.7 cells, murine macrophage cell line, were obtained from Korean Cell Line Bank (KCLB, Seoul, Korea) and seeded into a 96‐well plate (3 × 103 cells per well). After 1 day, the cells were treated with differentiation factors and each component of ZAB. After 5 days of treatment, the cells were fixed with 4% paraformaldehyde, rinsed with deionized water, and stained with a Takara TRAP stain kit. The stained samples were observed with optical microscopy. The cells were lysed using a Takara TRACP assay kit to quantify TRAP activity. The whole process was conducted following the provided protocol.
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