搜索

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   

下载 PDF 引用 收藏 提问与回复 分享您的反馈 Cited by

本文章节

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

  1. 2 ml microcentrifuge tubes (Eppendorf)
  2. Circular biopsy punch (ABC, catalog number: 0418 )
  3. 96-well microplates (Thermo Fisher Scientific, FisherbrandTM, catalog number: 21-377-203 )
  4. 3% Hydrogen peroxide (H2O2) (Sigma-Aldrich, catalog number: H1009 )
  5. 3, 3’-5, 5’-tetramethylbenzidine (TMB) (Sigma-Aldrich, catalog number: T2885 )
  6. 4-nitrophenyl-N-acetyl-β-D-glucosaminide (Sigma-Aldrich, catalog number: N9376 )
  7. Citric acid (Sigma-Aldrich, catalog number: 251275 )
  8. Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: 472301 )
  9. Glucose Solution (Sigma-Aldrich, catalog number: G8644 )
  10. Sulfuric acid (H2SO4) (Sigma-Aldrich, catalog number: 258105 )
  11. Hexadecyltrimethylammonium bromide (HTAB) (Sigma-Aldrich, catalog number: H9151 )
  12. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9541 )
  13. Potassium phosphate monobasic (KH2PO4) (Sigma-Aldrich, catalog number: P0662 )
  14. Liquid nitrogen
  15. Ethylenediaminetetraacetic acid disodium salt dihydrate (Na2EDTA) (Sigma-Aldrich, catalog number: V000114 )
  16. Sodium phosphate dibasic (Na2HPO4) (Sigma-Aldrich, catalog number: 255793 )
  17. Sodium phosphate tribasic dodecahydrate (Na3PO4) (Sigma-Aldrich, catalog number: 222003 )
  18. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S9888 )
  19. Sodium hydroxide (NaOH) (Sigma-Aldrich, catalog number: 221465 )
  20. Triton X-100 (Sigma-Aldrich, catalog number: T8787 )
  21. Phosphate buffered saline (PBS) (10x, pH 7.2) (see Recipes)
  22. PBS (1x) (see Recipes)
  23. Buffer 1 (see Recipes)
  24. Buffer 2 (see Recipes)
  25. H2O2 (0.002% v/v) (Sigma-Aldrich, catalog number: 7722-84-1 ) (see Recipes)
  26. MPO substrate solution (see Recipes)
  27. Saline-Triton buffer (see Recipes)
  28. Phosphate-citrate buffer (see Recipes)
  29. NAG substrate solution (see Recipes)
  30. Glycine buffer (see Recipes)

Equipment

  1. Freezer (-20 °C)
  2. Freezer (-80 °C)
  3. High Precision Laboratory Balance (Shimadzu Corporation, catalog number: ATX -224 )
  4. Micropipettes
  5. Microplate Spectrophotometer Reader (Molecular Devices, model: SpectraMax )
  6. Refrigerated centrifuge (Eppendorf, model: 5810R )
  7. Tissue homogenizer (Thermo Fisher Scientific, catalog number: PowerGen 125 )
  8. Vortex Mixer (VELP Scientifica, catalog number: F202A0171 )

Procedure

  1. Harvesting wound tissue
    1. The wounds are harvested after animal euthanasia. Anesthetic overdose is the preferred method for euthanasia. Avoid cervical dislocation or decapitation methods.
    2. First, remove the back skin of the mice and carefully remove the wound tissue with the aid of a circular biopsy punch (with a slightly larger diameter than the one employed to wounding) to collect entire wound area (plus about 2 mm surrounding skin tissue) (Figures 1A-D).
    3. Place the fresh samples (harvested tissue) in 2 ml microcentrifuge tubes and immediately dip in liquid nitrogen.
    4. Store the harvested tissue at -80 °C until use.
    5. To the excisional wound healing model, see Moreira et al. (2015).


      Figure 1. Harvesting wound tissues for analysis. After euthanasia, remove the dorsal wounded skin of mice (A). Place the larger circular punch over the skin lesion, press down, and slowly rotate to remove a circular piece of skin (B-C). Collect for NAG and MPO analyses (D) by immediately dipping the tissue-containing microcentrifuge tubes in liquid nitrogen.

  2. MPO enzyme isolation and assay (Figure 2)
    1. Weigh and homogenize the tissue in ice-cold Buffer 1 (100 mg tissue/1 ml buffer), using a powered high speed tissue homogenizer (Ultra-turrax or equivalent). Homogenizing procedures during enzyme isolation must be performed on ice.
    2. Centrifuge at 10,000 x g for 10 min at 4 °C.
    3. Discard supernatant and submit the pellet to hypotonic lysis as follows: Resuspend the pellet in 1 ml of 0.2% NaCl for 30 sec, and then add 1.0 ml of a 1.6% NaCl/5% glucose solution.
    4. Centrifuge at 10,000 x g for 10 min at 4 °C.
    5. Discard supernatant and resuspend the pellet in Buffer 2 (100 mg tissue/1 ml buffer).
    6. Vortex and then freeze-thaw three times using liquid nitrogen. The thawing step can be performed under running tap water.
    7. Centrifuged at 10,000 x g for 15 min at 4 °C.
    8. Save the supernatants for the enzymatic assay (if the MPO assay is not carried out immediately, the supernatants can be stored at -20 °C until used).
    9. Dilute samples in Buffer 2 at RT (we suggest 1:3, but the dilution should be adjusted experimentally).
    10. To assay, add 25 μl of diluted supernatants samples (or Buffer 2, used as blank) per well to 96-well microplate. All samples should be run in duplicate.
    11. Add 25 μl of MPO substrate solution.
    12. Incubate for 5 min at 37 °C.
    13. Add 25 μl of 0.002% H2O2.
    14. Incubate for 5 min at 37 °C.
    15. Finally, stop reaction with 25 μl of 1 M H2SO4.
    16. Read at 450 nm.
    17. Calculate the relative activity of MPO as follow: (sample absorbance spectrum - blank absorbance spectrum) x sample dilution factor. Express results as relative units (MPO activity/100 mg) that denote activity of MPO per 100 mg of tissue.


      Figure 2. MPO assay procedure summary

  3. NAG enzyme isolation and assay (Figure 3)
    1. Weigh and homogenize the tissue in ice-cold Saline-Triton buffer (100 mg tissue/1 ml buffer), using a powered high speed tissue homogenizer (Ultra-turrax or equivalent). Homogenizing procedures during enzyme isolation must be performed on ice.
    2. Centrifuge at 3,000 x g for 10 min at 4 °C.
    3. Save the supernatant for the enzymatic assay (if the NAG assay is not carried out immediately, the supernatants can be stored at -20 °C until used).
    4. Dilute assaying samples in phosphate-citrate buffer at RT (we suggest 1:3, but the dilution should be adjusted experimentally).
    5. To assay, add 25 μl of diluted supernatants samples (or phosphate-citrate buffer, used as blank) per well to 96-well microplate. All samples should be run in duplicate (Figure 4).
    6. Add 25 μl of NAG substrate solution.
    7. Incubate for 10 min at 37 °C.
    8. Finally, stop reaction with 25 μl of 0.2 M Glycine buffer.
    9. Read at 400 nm.
    10. Calculate the relative activity of NAG as follow: (Sample absorbance spectrum-blank absorbance spectrum) x sample dilution factor. Express results as relative units (NAG activity/100 mg) that denote activity of NAG per 100 mg of tissue.


      Figure 3. NAG assay procedure summary


      Figure 4. 96-Well NAG/MPO plate layout. Example of blank and sample distribution. All samples should be run in duplicate. For NAG, the blank is phosphate-citrate buffer and for MPO is buffer 2.

Notes

  1. The frozen supernatants saved for the enzymatic assays must be thawed at 4 °C and kept on ice during handling.
  2. Use a suitable ultra-permanent ink resistant pen that can resist to the severities of freezing and thawing at liquid nitrogen temperatures to properly identify the microcentrifuge tubes that will be used for the MPO enzyme isolation procedure.

Recipes

  1. Phosphate buffered saline (PBS) (10x, pH 7.2)
    NaCl 80 g (137 mM final concentration)
    Na2HPO4 11.05 g (or Na2HPO4.12H2O 29 g) (8.01 mM final concentration)
    KCl 2 g (2.7 mM final concentration)
    KH2PO4 2.1 g (1.5 mM final concentration)
    MilliQ water qsp 1,000 ml
  2. PBS (1x)
    PBS (10x) 50 ml
    MilliQ water 450 ml
  3. Buffer 1 (pH 4.7)
    NaCl 5.84 g (0.1 M final concentration)
    Na3PO4 3.12 g (0.02 M final concentration)
    Na2EDTA 5.58 g (add this only after pH adjustment) (0.015 M final concentration)
    MilliQ water qsp 1,000 ml
  4. Buffer 2 (pH 5.4)
    Na3PO4 7.8 g (0.05 M final concentration)
    HTAB 5 g (add only after pH adjustment) (0.5% p/v final concentration)
    MilliQ water qsp 1,000 ml
  5. H2O2 (2.4 mM final concentration)
    3% H2O2 7.0 μl
    Buffer 2 12 ml
  6. MPO substrate solution (1.6 mM final concentration; protect from light)
    TMB 3.845 mg
    DMSO 1 ml
  7. Saline-Triton buffer (Triton X-100 0.1% v/v final concentration)
    Saline solution (0.9%) - NaCl 9 g in MilliQ water qsp 1,000 ml
    Triton X-100 1 ml
  8. Phosphate-citrate buffer (pH 4.5)
    Solution A - Citric acid 9.6 g in MilliQ water qsp 500 ml (0.1 M final concentration)
    Solution B - Na2HPO4 17.9 g in MilliQ water qsp 500 ml (0.1 M final concentration)
    Mix 200 ml of solution A and 310 ml of solution B
  9. NAG Substrate solution (2.24 mM final concentration; protect from light)
    p-nitrophenyl-N-acetyl-β-D-glucosaminide 0.767 mg
    Phosphate-citrate buffer 1 ml
    Vortex and then sonicate the suspension for 10 sec
  10. Glycine buffer (0.2 M final concentration; pH 10.6)
    Solution A - Glycine 6.008 g in MilliQ water qsp 100 ml (0.8 M final concentration)
    Solution B - NaCl 4.68 g in MilliQ water qsp 100 ml (0.8 M final concentration)
    Solution C - NaOH 3.2 g in MilliQ water qsp 100 ml (0.8 M final concentration)
    Mix solutions A, B and C

Acknowledgments

This work was supported by Conselho Nacional de Pesquisa/CNPq, Coordenação de Aperfeiçoamento de Pessoal de Nível Superior/CAPES, Fundação de Amparo à Pesquisa de Minas Gerais/FAPEMIG, and Pró-reitoria de Graduação PROGRAD-UFMG, Brazil. CFM holds a PROBIC-FAPEMIG Scientific Initiation scholarship. PCV holds a CAPES PhD scholarship. MSF holds a PROGRAD-UFMG undergraduate scholarship. LSB holds a CNPq Research Fellowship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. We thank Maria Cecilia Campos Canesso and Thiago Bruno Rezende de Castro for technical support during the setting of the assays.

References

  1. Bailey, P. J. (1988). Sponge implants as models. Methods Enzymol 162: 327-334.
  2. Canesso, M. C., Vieira, A. T., Castro, T. B., Schirmer, B. G., Cisalpino, D., Martins, F. S., Rachid, M. A., Nicoli, J. R., Teixeira, M. M. and Barcelos, L. S. (2014). Skin wound healing is accelerated and scarless in the absence of commensal microbiota. J Immunol 193(10): 5171-5180.
  3. Moreira, C. F., Cassini-Vieira, P., da Silva, M. F. and Barcelos, L. S. (2015). Skin wound healing model - excisional wounding and assessment of lesion area. Bio-protocol 5(22): e1661.
  4. Kuebler, W. M., Abels, C., Schuerer, L. and Goetz, A. E. (1996). Measurement of neutrophil content in brain and lung tissue by a modified myeloperoxidase assay. Int J Microcirc Clin Exp 16(2): 89-97.

简介

炎症反应对于损伤后皮肤稳态的再建立是必需的。 在这种情况下,白细胞到达伤口部位并协调伤口愈合过程中的重要事件。 因此,对伤口组织中炎性细胞的特定亚群的定量是相当感兴趣的。 通过测量标记酶髓过氧化物酶(MPO)和N-乙酰基-β-D-氨基葡萄糖苷酶(NAG)的比活性,目前的方案集中于皮肤损伤中嗜中性粒细胞和巨噬细胞积累的定量指数。 MPO以高水平存在于嗜中性粒细胞和NAG的嗜苯胺粒细胞中,在激活的巨噬细胞的溶酶体中。 这些方法允许间接估计积累到皮肤中的嗜中性粒细胞和巨噬细胞的丰度。

材料和试剂

  1. 2ml微量离心管(Eppendorf)
  2. 圆形活检穿孔器(ABC,目录号:0418)
  3. 96孔微量培养板(Thermo Fisher Scientific,Fisherbrand TM ,目录号:21-377-203)
  4. 3%过氧化氢(H 2 O 2)(Sigma-Aldrich,目录号:H1009)
  5. 3,3'-5,5'-四甲基联苯胺(TMB)(Sigma-Aldrich,目录号:T2885)
  6. 4-硝基苯基-N-乙酰基-β-D-氨基葡糖苷(Sigma-Aldrich,目录号:N9376)
  7. 柠檬酸(Sigma-Aldrich,目录号:251275)
  8. 二甲基亚砜(DMSO)(Sigma-Aldrich,目录号:472301)
  9. 葡萄糖溶液(Sigma-Aldrich,目录号:G8644)
  10. 硫酸(H 2 SO 4)(Sigma-Aldrich,目录号:258105)
  11. 十六烷基三甲基溴化铵(HTAB)(Sigma-Aldrich,目录号:H9151)
  12. 氯化钾(KCl)(Sigma-Aldrich,目录号:P9541)
  13. 磷酸二氢钾(KH 2 PO 4)(Sigma-Aldrich,目录号:P0662)
  14. 液氮
  15. 乙二胺四乙酸二钠盐二水合物(Na 2 EDTA)(Sigma-Aldrich,目录号:V000114)
  16. 磷酸氢二钠(Na 2 HPO 4)(Sigma-Aldrich,目录号:255793)
  17. 磷酸三钠十二水合物(Na 3 PO 4)(Sigma-Aldrich,目录号:222003)
  18. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S9888)
  19. 氢氧化钠(NaOH)(Sigma-Aldrich,目录号:221465)
  20. Triton X-100(Sigma-Aldrich,目录号:T8787)
  21. 磷酸盐缓冲盐水(PBS)(10x,pH 7.2)(参见配方)
  22. PBS(1x)(请参阅配方)
  23. 缓冲区1(请参阅配方)
  24. 缓冲区2(参见配方)
  25. H 2 O 2(0.002%v/v)(Sigma-Aldrich,目录号:7722-84-1)(参见Recipes)
  26. MPO底物溶液(参见配方)
  27. 盐水 - Triton缓冲液(见配方)
  28. 磷酸盐 - 柠檬酸盐缓冲液(参见配方)
  29. NAG底物溶液(参见配方)
  30. 甘氨酸缓冲液(参见配方)

设备

  1. 冷冻(-20°C)
  2. 冷冻(-80℃)
  3. 高精度实验室天平(Shimadzu Coopration,目录号:ATX-224)
  4. 微量移液器
  5. 微孔板分光光度计读数器(Molecular Devices,型号:SpectraMax)
  6. 冷冻离心机(Eppendorf,型号:5810R)
  7. 组织匀浆器(Thermo Fisher Scientific,目录号:PowerGen 125)
  8. 涡旋混合器(VELP Scientifica,目录号:F202A0171)

程序

  1. 收获伤口组织
    1. 在动物安乐死后收获伤口。麻醉过量是 ?安乐死的首选方法。避免颈椎脱位 断头法。
    2. 首先,去除小鼠的背部皮肤 并借助于圆形活组织检查小心地除去伤口组织 冲头(具有比所采用的直径稍大的直径 伤口)以收集整个伤口区域(加上皮肤周围约2mm) ?组织)(图1A-D)
    3. 将新鲜样品(收获的组织)放在2ml微量离心管中,立即浸入液氮中
    4. 将收获的组织储存在-80℃直至使用
    5. 对于切除伤口愈合模型,参见Moreira等人(2015)。


      图1.收获伤口组织进行分析。安乐死后, 去除小鼠的背部受伤皮肤(A)。放置较大的圆形 冲击皮肤病变,按下,慢慢旋转除去a 圆形皮肤片(B-C)。收集NAG和MPO分析(D)由 立即将含有组织的微量离心管浸入 液氮。

  2. MPO酶分离和测定(图2)
    1. 在冰冷的缓冲液1(100 mg组织/1 ml)中称重并匀浆组织 ?缓冲液),使用动力高速组织匀浆器(Ultra-turrax或 ?当量)。酶分离过程中的匀浆程序必须 在冰上进行
    2. 在4℃下以10,000×g离心10分钟。
    3. 弃去上清液,将沉淀物置于低渗裂解液中 将沉淀重悬在1ml的0.2%NaCl中30秒,然后 加入1.0ml 1.6%NaCl/5%葡萄糖溶液
    4. 在4℃下以10,000×g离心10分钟。
    5. 弃去上清液,并将沉淀重悬于缓冲液2(100 mg组织/1 ml缓冲液)中
    6. 涡旋,然后使用液氮冻融三次。解冻步骤可以在自来水下进行。
    7. 在4℃下以10,000×g离心15分钟
    8. 保存上清液用于酶测定(如果MPO测定是 不立即进行,上清液可以在-20℃下保存 直到使用)。
    9. 在室温下稀释缓冲液2中的样品(我们建议1:3,但稀释应通过实验调整)
    10. 为了测定,加入25μl稀释的上清液样品(或缓冲液2, 用作空白)到96孔微量培养板中。所有样品应运行 ?一式两份
    11. 加入25μlMPO底物溶液
    12. 在37℃孵育5分钟。
    13. 加入25μl0.002%H 2 O 2 sub。
    14. 在37℃孵育5分钟。
    15. 最后,停止与25μl1M H 2 SO 4 4的反应。
    16. 在450 nm读取。
    17. 计算MPO的相对活性如下:(样品 吸收光谱 - 空白吸收光谱)x样品稀释 因子。以相对单位(MPO活性/100mg)表示结果 表示MPO每100mg组织的活性

      图2. MPO测定程序总结

  3. NAG酶分离和测定(图3)
    1. 在冰冷的Saline-Triton缓冲液(100 mg)中称取并匀浆组织 ?组织/1ml缓冲液),使用动力高速组织匀浆器 (Ultra-turrax或等同物)。酶的匀浆程序 必须在冰上进行隔离
    2. 在4℃下以3,000xg离心10分钟。
    3. 保存上清液用于酶测定(如果NAG测定是 不立即进行,上清液可以在-20℃下保存 直到使用)。
    4. 稀释测定样品在磷酸盐 - 柠檬酸盐缓冲液中 (我们建议1:3,但稀释度应该调整 实验)。
    5. 为了测定,加入25μl稀释的上清液 样品(或磷酸盐柠檬酸盐缓冲液,用作空白)加入96孔 ?微孔板。所有样品应重复运行(图4)。
    6. 加入25μlNAG底物溶液
    7. 在37℃孵育10分钟。
    8. 最后,停止反应与25μl的0.2M甘氨酸缓冲液。
    9. 在400 nm读取。
    10. 计算NAG的相对活性如下:(样品 吸收光谱 - 空白吸收光谱)x样品稀释因子。 ?表示作为相对单位(NAG活性/100mg)的结果 NAG每100mg组织的活性

      图3. NAG测定程序总结


      图4. 96孔NAG/MPO板布局。空白和样品的示例 分配。所有样品应重复运行。对于NAG,空白 ?是磷酸盐 - 柠檬酸盐缓冲液,对于MPO是缓冲液2。

笔记

  1. 保存用于酶测定的冷冻上清液必须在4℃下解冻,并在处理期间保持在冰上
  2. 使用合适的超永久耐墨笔,可以抵抗在液氮温度下冻结和解冻的严重性,以正确识别将用于MPO酶分离程序的微量离心管。

食谱

  1. 磷酸盐缓冲盐水(PBS)(10x,pH 7.2)
    NaCl 80g(最终浓度为137mM)
    Na 2 HPO 4 11.05g(或Na 2 HPO 4 sub)12.12g Na 2 HPO 4 11.05g(或Na 2 HPO 4) 2×29g)(8.01mM终浓度) KCl 2g(2.7mM终浓度) KH 2 PO 4 4 2.1g(最终浓度为1.5mM)
    MilliQ水qsp 1000 ml
  2. PBS(1x)
    PBS(10x)50ml
    MilliQ水450 ml
  3. 缓冲液1(pH 4.7)
    NaCl 5.84g(终浓度0.1μM) Na 3 PO 4 4.12g(0.02M最终浓度)
    Na 2 EDTA 5.58g(仅在pH调节后加入)(0.015M终浓度)
    MilliQ水qsp 1000 ml
  4. 缓冲液2(pH 5.4)
    (最终浓度为0.05M)的NH 3/Na 3 PO 4 4 HTAB 5g(仅在pH调节后加入)(0.5%p/v终浓度) MilliQ水qsp 1000 ml
  5. H 2 O 2(2.4mM终浓度) 3%H 2/2 O 27.0μl
    缓冲液2 12 ml
  6. MPO底物溶液(1.6mM终浓度;避光)
    TMB 3.845mg
    DMSO 1ml
  7. 盐水 - Triton缓冲液(Triton X-100 0.1%v/v终浓度) 盐水溶液(0.9%) - NaCl 9g在MilliQ水qsp 1000ml
    中 Triton X-100 1 ml
  8. 磷酸盐 - 柠檬酸盐缓冲液(pH 4.5)
    溶液A-柠檬酸9.6g在MilliQ水qsp 500ml(0.1M终浓度)中 溶液B-Na 2 HPO 4 17.9g在MilliQ水中qsp 500ml(0.1M终浓度)
    混合200ml溶液A和310ml溶液B
  9. NAG底物溶液(终浓度为2.24mM;避光)
    对硝基苯基-N-乙酰基-β-D-氨基葡萄糖苷0.767mg
    磷酸盐 - 柠檬酸盐缓冲液1ml
    涡旋,然后超声处理悬浮液10秒
  10. 甘氨酸缓冲液(0.2M终浓度; pH 10.6) 溶液A-甘氨酸6.008g在MilliQ水qsp100ml(0.8M终浓度)中 溶液B-NaCl 4.68g在MilliQ水qsp 100ml(0.8M终浓度)中 溶液C-NaOH 3.2g在MilliQ水qsp 100ml(0.8M终浓度)中 混合溶液A,B和C

致谢

这项工作得到了Consueho Nacional de Pesquisa/CNPq,Coordè??odeAperfei?oamentode Pessoal deNívelSuperior/CAPES,Funda??ode AmparoàPesquisa de Minas Gerais/FAPEMIG和Pró-reitoria deGradua??oPROGRAD-UFMG,Brazil的支持。 CFM拥有PROBIC-FAPEMIG科学启动奖学金。 PCV持有CAPES博士奖学金。无国界医生拥有PROGRAD-UFMG本科生奖学金。 LSB拥有CNPq研究奖学金。资助者在研究设计,数据收集和分析,决定发布或准备手稿方面没有任何作用。我们感谢Maria Cecilia Campos Canesso和Thiago Bruno Rezende de Castro在测定设置期间提供技术支持。

参考文献

  1. Bailey,P.J。(1988)。 海绵植入物作为模型。 方法Enzymol 162:327 -334。
  2. Canesso,M.C.,Vieira,A.T.,Castro,T.B.,Schirmer,B.G.,Cisalpino,D.,Martins,F.S.Rachid,M.A.,Nicoli,J.R.,Teixeira,M.M.and Barcelos,L.S.(2014)。 在没有共生微生物群的情况下,皮肤伤口愈合加速,无疤痕。 J Immunol 193(10):5171-5180
  3. Moreira,C.F.,Cassini-Vieira,P.,da Silva,M.F.and Barcelos,L.S。(2015)。 皮肤伤口愈合模型 - 切除伤口和病变面积评估生物方案< em> 5(22):e1661。
  4. Kuebler,W.M.,Abels,C.,Schuerer,L。和Goetz,A.E。(1996)。 通过改良的骨髓过氧化物酶测定法测量脑和肺组织中的嗜中性粒细胞含量。 Int J Microcirc Clin Exp 16(2):89-97。
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:Cassini-Vieira, P., Moreira, C. F., da Silva, M. F. and Barcelos, L. 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.
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。

当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。