Name: detecting migration and infiltration of neutrophils in mice
Uploaded: 2020-02-06T13:30:00
Description: Watch this Scientific Journal Video about Detecting Migration and Infiltration of Neutrophils in Mice at JoVE.com

这项工作得到了国家自然科学基金（助学金号81430099和31500704）、国际合作与交流项目（赠款号2014DFA32950）和北京中医药大学（捐赠）的支持。批号 BUCM-2019-JCRC006 和 2019-JYB-TD013）。

所有实验程序均经北京中医药大学动物护理与使用委员会审核通过。
注：使用C57BL/6小鼠（7-8周大）。
1. 中性粒细胞分离
<ol><li>围肠渗出细胞的采集<ol>
<li>在 ddH2O 中制备新鲜的 10% 蛋白酶肽溶液。根据小鼠数量计算所需的体积。 注：将小鼠数量设置为N，（2N+1）溶液的mL必须提前溶解和过滤。</li>
		<li>用 70% 乙醇喷洒工作空间。使用胰岛素注射...

<ol>
	<li>Klein, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+defects+in+severe+congenital+neutropenia:+emerging+insights+into+life+and+death+of+human+neutrophil+granulocytes.">Genetic defects in severe congenital neutropenia: emerging insights into life and death of human neutrophil granulocytes.</a> Annual Review of Immunology. 29, 399-413 (2011).</li><li>Nuzzi, P. A., Lokuta, M. A., Huttenlocher, A., Coutts, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+Neutrophil+Chemotaxis.">Analysis of Neutrophil Chemotaxis.</a> Adhesion Protein Protocols. , 23-35 (2007).</li><li>Margraf, A., Ley, K., Zarbock, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+Recruitment:+From+Model+Systems+to+Tissue-Specific+Patterns.">Neutrophil Recruitment: From Model Systems to Tissue-Specific Patterns.</a> Trends in Immunology. 40 (7), 613-634 (2019).</li><li>Bardoel, B. W., Kenny, E. F., Sollberger, G., Zychlinsky, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+balancing+act+of+neutrophils.">The balancing act of neutrophils.</a> Cell Host &amp; Microbe. 15 (5), 526-536 (2014).</li><li>Wright, H. L., Moots, R. J., Bucknall, R. C., Edwards, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+function+in+inflammation+and+inflammatory+diseases.">Neutrophil function in inflammation and inflammatory diseases.</a> Rheumatology. 49 (9), 1618-1631 (2010).</li><li>Thieblemont, N., Wright, H. L., Edwards, S. W., Witko-Sarsat, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+neutrophils+in+auto-immunity.">Human neutrophils in auto-immunity.</a> Seminars in Immunology. 28 (2), 159-173 (2016).</li><li>Oh, H., Siano, B., Diamond, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+isolation+protocol.">Neutrophil isolation protocol.</a> Journal of Visualized Experiments. (17), e745 (2008).</li><li>Filippi, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+transendothelial+migration:+updates+and+new+perspectives.">Neutrophil transendothelial migration: updates and new perspectives.</a> Blood. 133 (20), 2149-2158 (2019).</li><li>Kouspou, M. M., Price, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+cellular+migration+using+a+two-chamber+methodology.">Analysis of cellular migration using a two-chamber methodology.</a> Methods in Molecular Biology. 787, 303-317 (2011).</li><li>Muinonen-Martin, A. J., Veltman, D. M., Kalna, G., Insall, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+chamber+for+direct+visualisation+of+chemotaxis.">An improved chamber for direct visualisation of chemotaxis.</a> PLoS One. 5 (12), e15309 (2010).</li><li>Walheim, C. C., Zanin, J. P., de Bellard, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+trunk+neural+crest+cell+migration+using+a+modified+Zigmond+chamber+assay.">Analysis of trunk neural crest cell migration using a modified Zigmond chamber assay.</a> Journal of Visualized Experiments. (59), e3330 (2012).</li><li>Liew, P. X., Kubes, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Neutrophil's+Role+During+Health+and+Disease.">The Neutrophil's Role During Health and Disease.</a> Physiological Reviews. 99 (2), 1223-1248 (2019).</li><li>Ley, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophils:+New+insights+and+open+questions.">Neutrophils: New insights and open questions.</a> Science Immunology. 3 (30), eaat4579 (2018).</li><li>Tillack, K., Breiden, P., Martin, R., Sospedra, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+lymphocyte+priming+by+neutrophil+extracellular+traps+links+innate+and+adaptive+immune+responses.">T lymphocyte priming by neutrophil extracellular traps links innate and adaptive immune responses.</a> Journal of Immunology. 188 (7), 3150-3159 (2012).</li><li>Puga, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=B+cell-helper+neutrophils+stimulate+the+diversification+and+production+of+immunoglobulin+in+the+marginal+zone+of+the+spleen.">B cell-helper neutrophils stimulate the diversification and production of immunoglobulin in the marginal zone of the spleen.</a> Nature Immunology. 13 (2), 170-180 (2011).</li><li>Strzepa, A., Pritchard, K. A., Dittel, B. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myeloperoxidase:+A+new+player+in+autoimmunity.">Myeloperoxidase: A new player in autoimmunity.</a> Cellular Immunology. 317, 1-8 (2017).</li><li>Momohara, S., Kashiwazaki, S., Inoue, K., Saito, S., Nakagawa, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elastase+from+polymorphonuclear+leukocyte+in+articular+cartilage+and+synovial+fluids+of+patients+with+rheumatoid+arthritis.">Elastase from polymorphonuclear leukocyte in articular cartilage and synovial fluids of patients with rheumatoid arthritis.</a> Clinical Rheumatology. 16 (2), 133-140 (1997).</li><li>Swamydas, M., Luo, Y., Dorf, M. E., Lionakis, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+Mouse+Neutrophils.">Isolation of Mouse Neutrophils.</a> Current Protocols in Immunology. 110 (1), 3.20.1-3.20.15 (2015).</li><li>Luo, Y., Dorf, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+Mouse+Neutrophils.">Isolation of Mouse Neutrophils.</a> Current Protocols in Immunology. 22 (1), 3.20.1-3.20.6 (1997).</li><li>Carlson, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+neutrophil+lipocalin+is+a+unique+marker+of+neutrophil+inflammation+in+ulcerative+colitis+and+proctitis.">Human neutrophil lipocalin is a unique marker of neutrophil inflammation in ulcerative colitis and proctitis.</a> Gut. 50 (4), 501-506 (2002).</li><li>Nathan, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophils+and+immunity:+challenges+and+opportunities.">Neutrophils and immunity: challenges and opportunities.</a> Nature Reviews Immunology. 6 (3), 173-182 (2006).</li><li>Zhang, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tanshinone+IIA+ameliorates+chronic+arthritis+in+mice+by+modulating+neutrophil+activities.">Tanshinone IIA ameliorates chronic arthritis in mice by modulating neutrophil activities.</a> Clinical and Experimental Immunology. 190 (1), 29-39 (2017).</li><li>Forster, R., Sozzani, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+aspects+of+leukocyte+migration.">Emerging aspects of leukocyte migration.</a> European Journal of Immunology. 43 (6), 1404-1406 (2013).</li><li>Hu, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil-Mediated+Delivery+of+Dexamethasone+Palmitate-Loaded+Liposomes+Decorated+with+a+Sialic+Acid+Conjugate+for+Rheumatoid+Arthritis+Treatment.">Neutrophil-Mediated Delivery of Dexamethasone Palmitate-Loaded Liposomes Decorated with a Sialic Acid Conjugate for Rheumatoid Arthritis Treatment.</a> Pharmaceutical Research. 36 (7), 97 (2019).</li></ol>

从外周血7，骨髓和组织18高度纯化的中微粒细胞的详细方案已经提供很长时间。在这里，我们采用一种方法，将嗜中性粒细胞与围肠液19分离，其中成熟的嗜中性粒细胞保持灭活，以便进一步抗炎和抗氧化研究。
我们使用气囊实验来探索LPS诱导的嗜中性粒细胞在体内的渗透。该方法被提出作为直接测量细胞在体内一般炎症...

嗜中性粒细胞是最丰富的白细胞，占人类白细胞总体的50-70%。中性粒细胞是急性炎症的主要反应者之一。中性粒细胞可以通过组织驻留细胞2、3、4释放的细胞因子和化学因子的引导，被招募到炎症部位，由中性粒细胞表面的细胞粘附分子和血管内皮细胞5的相互作用来调节。中性粒细胞是宿主防御的基础，并在病理炎症反应中发挥作用，因为它们强大的能力通过释放活性氧物种（ROS）和其他破坏组织分子3，6来破坏组织。
先前的研究描述了小鼠或人类的几种中性粒细胞分离方案。Oh等人演示了一种密度梯度分离方法，将人类嗜中性粒细胞从全人类血液中分离出来7。然而，由于血量小，很难从小鼠血液中分离足够的嗜中性粒细胞。另外，可以从小鼠嗜虫液中引出大量纯和可行的小鼠中性粒细胞，这些纯化的嗜中性粒细胞可用于检查细胞外功能的几个方面，包括嗜中性粒细胞渗透、迁移、化学毒杆菌、氧化爆裂、细胞因子和中性粒细胞外陷阱（NET）生产8。Transwell测定9或Zigmond室测定10，11可用于评估嗜中性粒细胞在体外迁移。气囊模型用于评估嗜中性粒细胞在体内的迁移和渗透。皮下气囊模型是研究炎症细胞迁移的方便体内动物模型。
传统上，中性粒细胞被认为是炎症急性阶段的病原体消除剂。然而，最近的发现表明，嗜中性粒细胞是复杂的细胞，执行大量的特殊功能。嗜中性粒细胞可以调节许多过程，如急性损伤和修复，肿瘤发生，自身免疫反应，和慢性炎症12，13。中性粒细胞还调节适应性免疫反应，并能调节B细胞和T细胞14，15。中性粒细胞的大量短缺导致人类死亡或严重的免疫缺陷，小鼠的嗜中性粒细胞耗竭导致死亡，而器官中中性粒细胞的过度激活或招募会导致多种免疫疾病，如风湿性关节炎（RA）和系统性红斑狼疮（SLE）6。嗜中性粒细胞是RA患者的阴膜流体中最丰富的细胞。嗜中性粒细胞通过降解产生过量的骨髓过氧化物酶（MPO）和中性粒细胞酶（NE），从而加剧软骨侵蚀。MPO是一种过氧化物酶，主要表现在嗜中性粒细胞16的颗粒中。NE与关节软骨破坏有关17。MPO和NE可用于评估嗜中性粒细胞迁移和在RA患者组织中渗透的状态。
本文提供了三种常规方法，用于评估在体内和体外诱导的正常嗜中性粒细胞的迁移，以及病理性嗜中性粒细胞在小鼠关节特异性炎症模型中的渗透。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.1% Fast Green Solution</td>
			<td>Solarbio</td>
			<td>8348b</td>
			<td>Transfer 20 mg of fast grene FCF in one vial into another 100 mL beaker. Add 20 mL of H2O into the beaker and dissolve the stain by stirring to make 0.1% fast green solution, and filter it using a Nalgene PES 75mm filter</td>
		</tr>
		<tr>
			<td>0.1% Safranin O Staining Solution</td>
			<td>Solarbio</td>
			<td>8348a</td>
			<td>Transfer 20 mg of safranin O stain in one vial into a 100 ml beaker. Add 20 mL of H2O into the beaker and dissolve the stain by stirring to make 0.1% safranin O staining solution, and filter it using a Nalgene PES 75mm filter</td>
		</tr>
		<tr>
			<td>0.5 M Ethylenediaminetetraacetic acid solution (EDTA), pH 8.0</td>
			<td>Sigma</td>
			<td>324506</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>100% Ethanol</td>
			<td>Beijing Chemical Works</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>100% Methanol</td>
			<td>Beijing Chemical Works</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL Conical Polypropylene Centrifuge Tube</td>
			<td>Falcon</td>
			<td>14-959-53A</td>
			<td></td>
		</tr>
		<tr>
			<td>23 G x 1 1/4&#34; Needle</td>
			<td>BD</td>
			<td>305120</td>
			<td></td>
		</tr>
		<tr>
			<td>26 G x 3/8&#34; Needle</td>
			<td>BD</td>
			<td>305110</td>
			<td></td>
		</tr>
		<tr>
			<td>3% bovine serum albumin (BSA)</td>
			<td></td>
			<td></td>
			<td>Dissolve 0.3 g BSA in 10 mL PBS</td>
		</tr>
		<tr>
			<td>3% H2O2</td>
			<td></td>
			<td></td>
			<td>Mix 1 mL 30% H2O2 with methanol with 9 mL methanol</td>
		</tr>
		<tr>
			<td>3,3&#39;-diaminobenzidine (DAB) kit</td>
			<td>ZSGB-BIO</td>
			<td>ZLI-9018</td>
			<td></td>
		</tr>
		<tr>
			<td>30 G x 1/2&#34; Needle</td>
			<td>BD</td>
			<td>305106</td>
			<td></td>
		</tr>
		<tr>
			<td>30% H2O2</td>
			<td>Beijing Chemical Works</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL Syringe</td>
			<td>BD</td>
			<td>Z683574</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL Conical Polypropylene Centrifuge Tube</td>
			<td>Falcon</td>
			<td>14-432-22</td>
			<td></td>
		</tr>
		<tr>
			<td>50% Ethanol</td>
			<td></td>
			<td></td>
			<td>Mix 500 mL 100% ethanol with 500 mL dH2O</td>
		</tr>
		<tr>
			<td>54.8% Percoll</td>
			<td></td>
			<td></td>
			<td>Mix 2.74 mL SIP with 2.26 mL 1&#215;PBS, stand still</td>
		</tr>
		<tr>
			<td>70% Ethanol</td>
			<td></td>
			<td></td>
			<td>Mix 700 mL 100% ethanol with 300 mL dH2O</td>
		</tr>
		<tr>
			<td>70.2% Percoll</td>
			<td></td>
			<td></td>
			<td>Mix 3.51 mL SIP with 1.49 mL 1&#215;PBS, stand still</td>
		</tr>
		<tr>
			<td>80% Ethanol</td>
			<td></td>
			<td></td>
			<td>Mix 800 mL 100% ethanol with 200 mL dH2O</td>
		</tr>
		<tr>
			<td>95% Ethanol</td>
			<td></td>
			<td></td>
			<td>Mix 950 mL 100% ethanol with 50 mL dH2O</td>
		</tr>
		<tr>
			<td>Acid Alcohol Superfast Differentiation Solution</td>
			<td>Beyotime</td>
			<td>C0165S</td>
			<td></td>
		</tr>
		<tr>
			<td>ANTIBODIES</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Myeloperoxidase Antibody</td>
			<td>Abcam</td>
			<td>ab208670</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Neutrophil Elastase Antibody</td>
			<td>Abcam</td>
			<td>ab21595</td>
			<td></td>
		</tr>
		<tr>
			<td>Automatic Hematology Analyzer</td>
			<td>Sysmex</td>
			<td>XS-800i</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>VWR</td>
			<td>0332-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Complete Freund&#39;s Adjuvant, 10 mg/ml</td>
			<td>sigma</td>
			<td>1002036152</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover Slip</td>
			<td>CITOGLAS</td>
			<td>10212432C</td>
			<td></td>
		</tr>
		<tr>
			<td>Dial Thickness Gauge</td>
			<td>Mitutoyo</td>
			<td>7301</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf Microtubes, 1.5 mL</td>
			<td>Sigma</td>
			<td>Z606340</td>
			<td></td>
		</tr>
		<tr>
			<td>Foetal Bovine Serum (FBS) Premium</td>
			<td>PAN</td>
			<td>P30-1302</td>
			<td></td>
		</tr>
		<tr>
			<td>Gas Anesthesia System</td>
			<td>ZS Dichuang</td>
			<td>ZS-MV-IV</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat Anti-Rabbit IgG H&#38;L (HRP)</td>
			<td>PPLYGEN</td>
			<td>C1309</td>
			<td>This is the secondary antibody used in the immunohistochemical staining.</td>
		</tr>
		<tr>
			<td>Hank&#39;s Balanced Salt Solution (HBSS)</td>
			<td>Biological Industries, Beth HaEmek, Israel</td>
			<td>02-016-1A</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>Hematoxylin Staining Solution</td>
			<td>ZSGB-BIO</td>
			<td>ZLI-9609</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipopolysaccharide (LPS)</td>
			<td>Sigma</td>
			<td>L3012</td>
			<td></td>
		</tr>
		<tr>
			<td>MEDIA AND SUPPLEMENTS</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Modified Safranin O-fast Green FCF Cartilage Stain Kit</td>
			<td>Solarbio</td>
			<td>G1371</td>
			<td></td>
		</tr>
		<tr>
			<td>N-formyl-Met-Leu-Phe (fMLP)</td>
			<td>Sigma</td>
			<td>47729</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin Streptomycin Solution, 100&#215;</td>
			<td>Invitrogen</td>
			<td>1514022</td>
			<td></td>
		</tr>
		<tr>
			<td>Percoll</td>
			<td>GE Healthcare</td>
			<td>10245207</td>
			<td>Density gradient medium</td>
		</tr>
		<tr>
			<td>Permeabilization Buffer</td>
			<td></td>
			<td></td>
			<td>Mix 100 &#956;L Triton X-100 with 1 L dH2O to get 0.01% Triton X-100</td>
		</tr>
		<tr>
			<td>Phosphate Buffer Saline (PBS), 1&#215;</td>
			<td></td>
			<td></td>
			<td>Mix 90% ddH2O with 10% (v/v) 10&#215;PBS, autoclaved</td>
		</tr>
		<tr>
			<td>Phosphate Buffer Saline (PBS), 10&#215;</td>
			<td></td>
			<td></td>
			<td>Dissolve 16 g NaCl, 0.4 g KCl, 2.88 g Na2HPO4&#183;2H2O, 0.48 g KH2PO4 (anhydrous) in 200 mL ddH2O, adjust pH 7.4, autoclaved</td>
		</tr>
		<tr>
			<td>PLASTIC WARES AND EQUIPMENTS</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>POWDER</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Proteose Peptone</td>
			<td>Oxoid</td>
			<td>1865317</td>
			<td></td>
		</tr>
		<tr>
			<td>Retrieval Buffer</td>
			<td></td>
			<td></td>
			<td>Mix 18 mL retrieval buffer A with 82 mL retrieval buffer B, add dH2O to 1000 mL, adjust pH to 6.0</td>
		</tr>
		<tr>
			<td>Retrieval Buffer A Stock for IHC</td>
			<td></td>
			<td></td>
			<td>Dissolve 4.2 g citric acid (C6H5O7&#183;H2O) in 200 mL dH2O</td>
		</tr>
		<tr>
			<td>Retrieval Buffer B Stock for IHC</td>
			<td></td>
			<td></td>
			<td>Dissolve 5.88 g trisodium citrate dihydrate (C6H5Na3O7&#183;2H20) in 200 mL dH2O</td>
		</tr>
		<tr>
			<td>Roswell Park Memorial Institute (RPMI)-1640 medium</td>
			<td>Sigma</td>
			<td>R8758</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI-1640 Complete Medium</td>
			<td></td>
			<td></td>
			<td>RPMI-1640 medium is supplemented with 10% FBS and 1% penicillin/streptomycin.</td>
		</tr>
		<tr>
			<td>Shu Rui U40 Disposable Sterile Insulin Injection Needle 1 mL</td>
			<td>BD</td>
			<td>328421</td>
			<td></td>
		</tr>
		<tr>
			<td>Slide</td>
			<td>CITOGLAS</td>
			<td>10127105P-G</td>
			<td></td>
		</tr>
		<tr>
			<td>SOLUTION</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Stock Isotonic Percoll (SIP)</td>
			<td></td>
			<td></td>
			<td>Mix 90% (v/v) of percoll with 10% (v/v) 10&#215;PBS, stand still for 20 min</td>
		</tr>
		<tr>
			<td>Wash Buffer in Air Pouch Assay</td>
			<td></td>
			<td></td>
			<td>Dilute 0.5M EDTA to 10mM with HBSS</td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>Beijing Chemical Works</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

detecting migration and infiltration of neutrophils in mice

嗜中性粒细胞是先天免疫系统的主要成员，在宿主防御病原体和病理炎症反应方面发挥着关键作用。中性粒细胞可以通过细胞因子和化学因子的指导被招募到炎症部位。中性粒细胞的渗透可能导致不分青红皂白的组织损伤，如风湿性关节炎（RA）。从围肠渗出物分离的嗜中性粒细胞对定义的化学吸引剂N-form-Met-Leu-Phe（fMLP）有反应，在Transwell或Zigmond室测定中体外。气囊实验可用于评估中性粒细胞对体内脂多糖（LPS）的化学性评价。佐剂诱发关节炎（AA）小鼠模型常用于RA研究，与抗骨髓过氧化物酶（MPO）或抗中性粒细胞酶（NE）抗体的关节部分的免疫组织化学染色是一种公认的测量方法中性粒细胞渗透。这些方法可用于发现针对中性粒细胞迁移的有前途的疗法。

从小鼠的洗漱液中收集围渗出细胞。细胞在 1 mL 的 RPMI-1640 完整介质中重新悬浮，分层到两步 （54.8%/70.2%）不连续密度梯度（图1A），在1，500 x g下离心30分钟。 嗜中性粒细胞（+95%，±1 x 107嗜中性粒细胞/小鼠）从下界面中恢复（图1B）。
进行?...

Watch this Scientific Journal Video about Detecting Migration and Infiltration of Neutrophils in Mice at JoVE.com

Detecting Migration and Infiltration of Neutrophils in Mice

在这里，我们提出了三种方法，以评估嗜中性粒细胞迁移和渗透在体内和体外。这些方法可用于发现针对中性粒细胞迁移的有前途的治疗方法。

检测小鼠中性粒细胞的迁移和渗透

Neutrophils are a major member of the innate immune system and play pivotal roles in host defense against pathogens and pathologic inflammatory reactions. ...

该方法试图描述三种常用的方法，用于评估正常嗜中性粒细胞在体内和体外迁移，以及病理嗜中性粒细胞在小鼠关节炎模型中的渗透。我们认为这种方法对于该领域的其他研究人员可能有用。该协议包含的方法细节，以评估嗜中性粒细胞在炎症点使用空气袋测定和辅助诱导关节炎的迁移能力。通过将模型组与治疗组进行比较，该技术对关节炎治疗的影响延伸到了关节炎治疗中。这种方法可以提供炎症性疾病的见解，并可应用于炎症性肠病。演示程序将是清一路和海旭江，研究生从我们实验室。在零日对小鼠进行麻醉后，使用连接到五毫升注射器的0.22微米过滤器获得三毫升的灭菌空气。用钳子抬起麻醉小鼠的后皮，用 26 量表 3/8 英寸针注射三毫升灭菌空气。治疗后，将小鼠从呼吸装置中取出，并放入井架。监测小鼠，以确保它们还活着，直到它们开始四处走动。第三天，将另外三毫升的消毒空气注入先前建立的气囊，以维持空气袋。第六天，将不同的治疗注射到空气袋中。注射一毫升PBS作为负对照，并注射每毫升LPS一毫升一微克作为正对照诱导局部炎症。六个小时后，牺牲老鼠。对于每个空气袋，注射一毫升的洗涤缓冲液来清洗空气袋，并在15毫升离心管中收集炎症的出水液。然后用两毫升的洗涤缓冲液清洗空气袋两次，并在同一离心管中收集炎症的排泄液。在室温下以100倍g离心10分钟。丢弃上流液，将细胞重新在一毫升的洗涤缓冲液中。使用自动血液学分析仪计算细胞数值以量化嗜中性粒细胞比率。通过旋转至少五秒钟，将 100 微升悬浮液放入胰岛素注射器中，暂停完成 Freund 的辅助剂。麻醉后，标记所选的爪子，从喷油器将 20 微升完整的 Freund 辅助剂注入脚踝关节空间的四个近侧点。将加工过的老鼠放入新房间。监测小鼠，以确保它们呼吸，直到他们重新获得移动能力。每三天，使用袖珍厚度计测量脚踝关节直径。此外，评估关节炎的严重程度，以关节炎评分标准。零是正常的。没有红斑和肿胀的证据。一种是最轻微的关节炎。Erythema 和轻度肿胀仅限于柏油或脚踝关节。二是中度关节炎。Erythema 和轻度肿胀从脚踝延伸到焦油。三是严重的关节炎。Erythema 和中度肿胀从脚踝延伸到骨质关节。四是最严重的关节炎。红斑和严重的肿胀包括脚踝，脚和数字，或肢体的骨病。牺牲鼠标后，用钳子和剪刀从后腿取出皮肤和部分肌肉。用70%乙醇喷洒关节，用纸巾去除其余肌肉。在室温下将脚踝关节固定为4%甲醛两天。然后在室温下将接头在 10%EDTA 中脱花一个月，并每周更换介质。一个月后，将组织放在一个标记的模具中，在大约60摄氏度的液体石蜡中嵌入组织。简单地冷却。将微原子上的厚度设置为四微米并切割切片。然后将部分漂浮在43摄氏度的水浴中，以在短时间内扩大部分。将部分安装到幻灯片上，并在 70 摄氏度下将幻灯片放入烤箱两小时。为将来使用，请将幻灯片保存在零下 20 摄氏度。接下来，将幻灯片放在机架中，根据手稿对二甲苯和乙醇进行洗涤，在室温下进行补水。最后，用自来水洗三分钟。然后在0.1%的快速绿色溶液中染色5分钟。用1%醋酸冲洗10秒，用0.1%的Safranin O染色溶液染色20分钟。之后，根据手稿将幻灯片浸入二甲苯和乙醇洗涤解决方案中。将组织部分安装到对象阶段，并在显微镜下观察组织。要可视化嗜中性粒细胞，首先在 78 摄氏度下烘烤石蜡部分两个小时，进行免疫组织化学染色。将幻灯片放在机架中，根据手稿执行二甲苯、乙醇、水和 PBS 洗涤，在室温下进行补水。然后加入一滴渗透缓冲液覆盖组织。在37摄氏度的湿度控制托盘中孵育部分10分钟。之后，在 PBS 中冲洗幻灯片三分钟三次。避免直接冲洗组织。接下来，执行热诱导抗原表位检索。将幻灯片排列在机架中。将幻灯片浸入充满检索缓冲器的压力锅炉中。将压力锅炉放入微波炉中，将微波炉设置为 600 瓦，加热幻灯片 10 分钟。煮沸后，将锅炉中的滑梯冷却至90摄氏度。拿出幻灯片，在 PBS 中冲洗三分钟三次。要淬火内源过氧化物酶活性，将幻灯片浸入新鲜准备的3%过氧化氢室温下15分钟。在 PBS 中冲洗幻灯片三分钟三次。用疏水笔在样品周围勾勒出一个大圆圈。避免接触样品。将 50 至 100 微升的 3%牛血清白蛋白添加到样品中，将样品在 37 摄氏度的湿度控制室中阻挡并放置 60 分钟。然后删除阻塞解决方案。快速将50微升PBS稀释原抗体加入到每个部分。在四摄氏度的湿度控制托盘中孵育滑梯。第二天，拿出托盘，让它在室温下站立30分钟。然后在 PBS 中冲洗幻灯片三分钟三次。向组织添加50微升PBS稀释的二级抗体。在37摄氏度的湿度控制托盘中孵育滑梯30分钟。然后在 PBS 中冲洗幻灯片三分钟三次。在稀释的DAB溶液中开发五分钟。避免因民建联反应过度而出现深色。用蒸馏水冲洗滑梯。将幻灯片用赤氧林中计数器 10 秒钟，然后用自来水冲洗幻灯片 5 分钟。在酸性酒精中超快的分化溶液中冲洗三秒钟，然后在自来水中冲洗10分钟。根据手稿，将幻灯片浸入乙醇和二甲苯的室温下清洗。使用安装解决方案固定盖玻片。在显微镜下观察组织。在这项研究中，进行了空气袋实验，以调查体内LPS刺激的嗜中性粒细胞招募。空气袋中出出的白细胞子集比控制中的白细胞子集高得多。与对照组相比，辅助性诱发关节炎组在爪子上表现出显著的水肿。脚踝关节直径增加，关节炎评分持续上升。软骨损伤是类风湿性关节炎的代表性综合征。CFA的挑战导致大量的白细胞渗透，严重的软骨侵蚀，和突触增生。任何表达水平的MPO都是中性粒细胞渗透的代表性标志物，在关节部分被明显调节。确保空气被注入皮肤，而不是注入肌肉。我们也可以研究通过免疫组织化学染色的脚踝关节组织部分与抗p84或这些方法可用于研究其他炎症性疾病，如炎症性肠病的中性粒细胞外陷阱的形成。

Research

JoVE Journal

Biology

Radioactive in situ Hybridization for Detecting Diverse Gene Expression Patterns in Tissue

Radioactive in situ Hybridization for Detecting Diverse Gene Expression Patterns in Tissue

放射性的原位杂交检测组织不同的基因表达模式

Knowing the timing, level, cellular localization, and cell type that a gene is expressed in contributes to our understanding of the function of the gene. ...

科研

生物学

Detecting Somatic Genetic Alterations in Tumor Specimens by Exon Capture and Massively Parallel Sequencing

由外显子捕获和大规模平行测序检测体细胞遗传学改变在肿瘤标本

Efforts  to detect and investigate key oncogenic mutations have proven valuable to  facilitate the appropriate treatment for cancer patients. The ...

Detecting, Visualizing and Quantitating the Generation of Reactive Oxygen Species in an Amoeba Model System

检测，可视化和定量活性氧的生成在变形虫模型系统

Reactive oxygen species (ROS) comprise a range of reactive and short-lived, oxygen-containing molecules, which are dynamically interconverted or ...

In vitro Cell Migration and Invasion Assays

In vitro Cell Migration and Invasion Assays

在体外细胞迁移和侵袭实验

Migration is a key property of live cells and critical for normal development, immune response, and disease processes such as cancer metastasis and ...

Imaging Intermediate Filaments and Microtubules with 2-dimensional Direct Stochastic Optical Reconstruction Microscopy

2维直接随机光学重建显微成像中间丝和微管

The cytoskeleton, composed of actin microfilaments, microtubules, and intermediate filaments (IF), plays a key role in the control of cell shape, ...

The MUB40 Peptide for Use in Detecting Neutrophil-Mediated Inflammation Events

The MUB40 Peptide for Use in Detecting Neutrophil-Mediated Inflammation Events

mub40肽在中性炎症事件检测中的应用

Here, we provide a protocol involving the use of MUB40, a synthesized peptide with the ability to bind glycosylated lactoferrin stored at high ...

Detecting and Characterizing Protein Self-Assembly In Vivo by Flow Cytometry

通过流动细胞测定检测和描述 Vivo 中的蛋白质自组装

Protein self-assembly governs protein function and compartmentalizes cellular processes in space and time. Current methods to study it suffer from ...

Detecting Establishment of Shared Blood Supply in Parabiotic Mice by Caudal Vein Glucose Injection

考达尔静脉葡萄糖注射剂在益生菌小鼠中检测共享血液供应的建立

Parabiosis is an experimental method for surgically combining two parallel animals along the longitudinal axis of the body. We present a protocol for ...

A Cost Effective and Adaptable Scratch Migration Assay

经济高效且适应性强的划痕迁移检测

Cell migration is a key component in both physiological and pathological events. Normal cell migration is required for essential functions such as ...

Isolation of Human Neutrophils from Whole Blood and Buffy Coats

从全血和巴菲皮脂中分离出人类嗜中性粒细胞

Neutrophils (PMNs) are the most abundant leukocytes in human circulation, ranging from 40 to 70% of total blood leukocytes. They are the first cells ...