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 |
Tags
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).