Pit Assay to Measure the Bone Resorptive Activity of Bone Marrow-derived Osteoclasts
陷孔试验测量骨髓源破骨细胞的骨吸收活性
发布: 2016年06月20日第6卷第12期 DOI: 10.21769/BioProtoc.1836 浏览次数: 14529
评审: Jalaj GuptaAnonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
May 2015
Abstract
Although it is possible to use a tartrate-resistant acid phosphatase (TRAP) stain to assist in identifying osteoclasts, a separate method is needed to determine the bone resorption activity of osteoclasts. Since osteoclasts leave “pits” after bone matrix resorption (Charles et al., 2014), it is possible to stain pits as a method of measuring osteoclast bone resorption activity. The pit assay protocol enables researchers to stain bony slices that were co-cultured with osteoclasts with toluidine blue in order to allow the visualization, capture, and analysis of osteoclast resorptive activity based on the number, size and depth of pits (Zhou et al., 2015). The pit assay protocol is separated into three sequential stages: Preparation of bone slices (1); preparation of osteoclast precursors (Ross et al., 2006; Teitelbaum et al., 2000) (2), and bone resorption pit assay (3).
Keywords: Osteoclast function (破骨细胞功能)
Bone resorption (骨吸收)
Pit assay (坑试验)
Materials and Reagents
- Parafilm
- Razor blade
- Tissue culture tubes (standard 15 or 50 ml conical TC tubes)
- 5 ml syringe with 25 G needle
- Cell strainer (70 μm) (Corning, catalog number: 352350 )
- Cell culture 96 well plates
- Tissue culture petri dish
- Whatman filter paper (Sigma-Aldrich, catalog number: WHA10347509 )
- 6- to 10-week-old mice
- Phosphate buffered saline (PBS), sterilized, pH 7.4 (Thermo Fisher Scientific, catalog number: 10010023 )
- Dulbecco's modified Eagle medium, high glucose (Thermo Fisher Scientific, GibcoTM, catalog number: 11965092 )
- Penicillin/streptomycin (10,000 U/ml) (Thermo Fisher Scientific, catalog number: 15140-122 )
- Fetal bovine serum (FBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 16000044 )
- MEMα, nucleosides (Thermo Fisher Scientific, catalog number: 12571-063 )
- Macrophage-stimulating factor (M-CSF) (PeproTech, catalog number: 315-02 ) and receptor activated nuclear factor k-B ligand (RANKL) (PeproTech, catalog number: 315-11 )
- Bovine femurs (from meat market)
- Ethanol
- Glutaraldehyde (Sigma-Aldrich, catalog number: G5882 )
- Toluidine blue (Sigma-Aldrich, catalog number: T3260-5G )
- MilliQ water
- Acid phosphatase, leukocyte (TRAP) Kit (Sigma-Aldrich, catalog number: 387A-1KT )
- Sodium borate 10-hydrate (Fisher Scientific, catalog number: 02-003-999 )
- Trypan-blue (Thermo Fisher Scientific, catalog number: T6146-5G )
- Ammonium chloride (NH4Cl) (Sigma-Aldrich, catalog number: A9434-500G )
- Trizma® hydrochloride (Tris-HCl) (Sigma-Aldrich, catalog number: T5941-100G )
- Red blood cell (RBC) lysis buffer (Sigma-Aldrich, catalog number: R7757 ) (see Recipes)
- Osteoclast precursor culture media (see Recipes)
- Osteoclast differentiation media (see Recipes)
- 1% toluidine blue (see Recipes)
- Glutaraldehyde solution (see Recipes)
Equipment
- Forceps scissors
- Hacksaw
- Scalpel
- Water ultrasonicator
- Isomet low speed saw (BUEHLER, catalog number: 111280 ) with wafering blade
- Light microscope
Procedure
文章信息
版权信息
© 2016 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Vesprey, A. and Yang, W. (2016). Pit Assay to Measure the Bone Resorptive Activity of Bone Marrow-derived Osteoclasts. Bio-protocol 6(12): e1836. DOI: 10.21769/BioProtoc.1836.
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分类
干细胞 > 成体干细胞 > 维持和分化
细胞生物学 > 细胞染色 > 全细胞
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