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

Published: April 30, 2023

Abstract

Source: Dai, Q., et al. A RANKL-based Osteoclast Culture Assay of Mouse Bone Marrow to Investigate the Role of mTORC1 in Osteoclast Formation. J. Vis. Exp. (2018).

This video demonstrates the tartrate-resistant acid phosphatase staining technique to detect the presence of TRAP-containing granules inside osteoclasts.  The technique forms magenta-colored dye granule deposits in the cytoplasm of osteoclasts, rendering them visible under an inverted microscope.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board. 1. Preparation Generate osteoclast specific Raptor deletion mice (Raptorfl/fl; Ctsk-cre, hereafter RapCtsk) by mating Raptorfl/fl mice with Ctsk-cre mice. Use the Raptorfl/fl</e…

Representative Results

Figure 1: Representative view of osteoclasts. (A) Giant osteoclasts observed in bright field on day 6. The red line outlined the border of osteoclasts. (B) A typically giant, multinucleated, TRAP positive (wine red) osteoclast. The black arrow indicated two nuclei of the multinucleated osteoclast.

Divulgazioni

The authors have nothing to disclose.

Materials

Raptorfl/fl mice The Jackson Laboratory 13188
Ctsk-cre mice a gift from S. Kato, University of Tokyo, Tokyo, Japan
α-MEM Corning 10-022-CVR
Glutamine Gibico 25030081
Penicillin streptomycin Gibico 15140122
Fetal calf serum BioInd 04-001-1A
Recombinant mouse M-CSF protein R&D Q3U4F9
Recombinant mouse RANKL protein R&D Q3TWY5
RBC lysis buffer Beyotime C3702
Trypan blue Sigma-Aldrich 302643
Acetone Shanghai Chemical Co. Ltd.
Citrate solution Sigma-Aldrich 915
Formaldehyde solution Shanghai Chemical Co. Ltd.
Acid Phosphatase, Leukocyte (TRAP) Kit Sigma-Aldrich 387A-1KT
Fast Garnet GBC Base solution Sigma-Aldrich 3872
Sodium Nitrite Solution Sigma-Aldrich 914
Naphthol AS-BI Phosphate Solution Sigma-Aldrich 3871
Acetate solution Sigma-Aldrich 3863
Tartrate solution Sigma-Aldrich 3873
Dulbecco's phosphate-buffered saline Corning 21-031-CVR
37% formaldehyde Xilong scientific
IX71 Olympus
0.45-mm Syringe
Scissor
Mosquito forcep

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Citazione di questo articolo
Tartrate-Resistant Acid Phosphatase Staining: An In Vitro Technique to Detect TRAP Enzyme-Containing Cultured Osteoclasts. J. Vis. Exp. (Pending Publication), e21047, doi: (2023).

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