Estimation of Wound Tissue Neutrophil and Macrophage Accumulation by Measuring Myeloperoxidase (MPO) and N-Acetyl-β-D-glucosaminidase (NAG) Activities
通过测定髓过氧化物酶（MPO）和N-乙酰-β-D-氨基葡萄糖苷酶（NAG）的活性估计创伤组织的嗜中性白细胞和巨噬细胞的积聚
发布: 2015年11月20日第5卷第22期 DOI: 10.21769/BioProtoc.1662 浏览次数: 14341
评审: Ivan ZanoniAchille BroggiMarco Di Gioia
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参见作者原研究论文
The authors used this protocol in:
Nov 2014
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Abstract
The inflammatory response is essential to the reestablishment of cutaneous homeostasis following injury. In this context, leukocytes arrive at the wound site and orchestrate essential events in the wound healing process. Therefore, the quantification of specific subsets of inflammatory cells in the wound tissue is of considerable interest. The current protocol focus on a quantitative index of neutrophils and macrophages accumulation within skin lesions by measuring the specific activity of the marker enzymes Myeloperoxidase (MPO) and N-acetyl-β-D-glucosaminidase (NAG), respectively. MPO is present in high levels in the azurophilic granules of neutrophils and NAG in lysosomes of activated macrophages. These methods allow the indirect estimation of the abundance of neutrophils and macrophages accumulated into the skin.
Materials and Reagents
- 2 ml microcentrifuge tubes (Eppendorf)
- Circular biopsy punch (ABC, catalog number: 0418 )
- 96-well microplates (Thermo Fisher Scientific, FisherbrandTM, catalog number: 21-377-203 )
- 3% Hydrogen peroxide (H2O2) (Sigma-Aldrich, catalog number: H1009 )
- 3, 3’-5, 5’-tetramethylbenzidine (TMB) (Sigma-Aldrich, catalog number: T2885 )
- 4-nitrophenyl-N-acetyl-β-D-glucosaminide (Sigma-Aldrich, catalog number: N9376 )
- Citric acid (Sigma-Aldrich, catalog number: 251275 )
- Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: 472301 )
- Glucose Solution (Sigma-Aldrich, catalog number: G8644 )
- Sulfuric acid (H2SO4) (Sigma-Aldrich, catalog number: 258105 )
- Hexadecyltrimethylammonium bromide (HTAB) (Sigma-Aldrich, catalog number: H9151 )
- Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9541 )
- Potassium phosphate monobasic (KH2PO4) (Sigma-Aldrich, catalog number: P0662 )
- Liquid nitrogen
- Ethylenediaminetetraacetic acid disodium salt dihydrate (Na2EDTA) (Sigma-Aldrich, catalog number: V000114 )
- Sodium phosphate dibasic (Na2HPO4) (Sigma-Aldrich, catalog number: 255793 )
- Sodium phosphate tribasic dodecahydrate (Na3PO4) (Sigma-Aldrich, catalog number: 222003 )
- Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S9888 )
- Sodium hydroxide (NaOH) (Sigma-Aldrich, catalog number: 221465 )
- Triton X-100 (Sigma-Aldrich, catalog number: T8787 )
- Phosphate buffered saline (PBS) (10x, pH 7.2) (see Recipes)
- PBS (1x) (see Recipes)
- Buffer 1 (see Recipes)
- Buffer 2 (see Recipes)
- H2O2 (0.002% v/v) (Sigma-Aldrich, catalog number: 7722-84-1 ) (see Recipes)
- MPO substrate solution (see Recipes)
- Saline-Triton buffer (see Recipes)
- Phosphate-citrate buffer (see Recipes)
- NAG substrate solution (see Recipes)
- Glycine buffer (see Recipes)
Equipment
- Freezer (-20 °C)
- Freezer (-80 °C)
- High Precision Laboratory Balance (Shimadzu Corporation, catalog number: ATX -224 )
- Micropipettes
- Microplate Spectrophotometer Reader (Molecular Devices, model: SpectraMax )
- Refrigerated centrifuge (Eppendorf, model: 5810R )
- Tissue homogenizer (Thermo Fisher Scientific, catalog number: PowerGen 125 )
- Vortex Mixer (VELP Scientifica, catalog number: F202A0171 )
Procedure
文章信息
版权信息
© 2015 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Cassini-Vieira, P., Moreira, C. F., da Silva, M. F. and Barcelos, L. D. S. (2015). Estimation of Wound Tissue Neutrophil and Macrophage Accumulation by Measuring Myeloperoxidase (MPO) and N-Acetyl-β-D-glucosaminidase (NAG) Activities. Bio-protocol 5(22): e1662. DOI: 10.21769/BioProtoc.1662.
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分类
免疫学 > 免疫细胞功能 > 巨噬细胞
免疫学 > 免疫细胞功能 > 嗜中性粒细胞
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