Name: functional characterization of regulatory macrophages that inhibit graft-reactive immunity
Uploaded: 2017-06-07T12:30:00
Description: Watch this Scientific Journal Video about Functional Characterization of Regulatory Macrophages That Inhibit Graft-reactive Immunity at JoVE.com

我们承认西奈山流行细胞计数，显微外科学和生物储存库/病理学研究中心卓越的技术贡献。这项工作得到了COST行动BM1305：重点关注和加速细胞耐受性治疗的行动（FACTT），西奈半导体/米勒移植研究所发展基金，Ministry of de Ciencia e Innovacion SAF2013-48834-R和SAF2016-80031-R JO

<html>在这项研究中，小鼠按照美国农业部的指导方针和“实验动物护理和使用公共卫生服务指南”的建议。涉及动物使用的所有实验技术均按照西奈山医学院的制度动物护理和利用委员会（IACUC）批准的方案进行。 媒体准备<ol><li>通过使用2mM L-谷氨酰胺，1mM丙酮酸钠，0.1mM非必需氨基酸，5mM HEPES和0.05mM 2，制备具有10％FBS和1％青霉素 - 链霉素（10,000U / mL）的完整RPMI 1640培养基?...

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	<li>Jose, M. D., Ikezumi, Y., van Rooijen, N., Atkins, R. C., Chadban, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophages+act+as+effectors+of+tissue+damage+in+acute+renal+allograft+rejection.">Macrophages act as effectors of tissue damage in acute renal allograft rejection.</a> Transplantation. 76 (7), 1015-1022 (2003).</li><li>Oberbarnscheidt, M. H., 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=Non-self+recognition+by+monocytes+initiates+allograft+rejection.">Non-self recognition by monocytes initiates allograft rejection.</a> J Clin Invest. 124 (8), 3579-3589 (2014).</li><li>Garcia, M. 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D., 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=Optimized+staining+and+proliferation+modeling+methods+for+cell+division+monitoring+using+cell+tracking+dyes.">Optimized staining and proliferation modeling methods for cell division monitoring using cell tracking dyes.</a> J Vis Exp. (70), e4287 (2012).</li><li>Poujol, F., 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=Flow+cytometric+evaluation+of+lymphocyte+transformation+test+based+on+5-ethynyl-2'deoxyuridine+incorporation+as+a+clinical+alternative+to+tritiated+thymidine+uptake+measurement.">Flow cytometric evaluation of lymphocyte transformation test based on 5-ethynyl-2'deoxyuridine incorporation as a clinical alternative to tritiated thymidine uptake measurement.</a> J Immunol Methods. 415, 71-79 (2014).</li><li>Last'ovicka, J., Budinsky, V., Spisek, R., Bartunkova, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+lymphocyte+proliferation:+CFSE+kills+dividing+cells+and+modulates+expression+of+activation+markers.">Assessment of lymphocyte proliferation: CFSE kills dividing cells and modulates expression of activation markers.</a> Cell Immunol. 256 (1-2), 79-85 (2009).</li><li>Corry, R. 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(6), e238 (2007).</li><li>Ochando, J., Conde, P., Bronte, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monocyte-Derived+Suppressor+Cells+in+Transplantation.">Monocyte-Derived Suppressor Cells in Transplantation.</a> Curr Transplant Rep. 2 (2), 176-183 (2015).</li><li>Gallina, G., 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=Tumors+induce+a+subset+of+inflammatory+monocytes+with+immunosuppressive+activity+on+CD8++T+cells.">Tumors induce a subset of inflammatory monocytes with immunosuppressive activity on CD8+ T cells.</a> J Clin Invest. 116 (10), 2777-2790 (2006).</li><li>Huang, B., 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=Gr-1+CD115++immature+myeloid+suppressor+cells+mediate+the+development+of+tumor-induced+T+regulatory+cells+and+T-cell+anergy+in+tumor-bearing+host.">Gr-1+CD115+ immature myeloid suppressor cells mediate the development of tumor-induced T regulatory cells and T-cell anergy in tumor-bearing host.</a> Cancer Res. 66 (2), 1123-1131 (2006).</li><li>Luan, Y., 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=Monocytic+myeloid-derived+suppressor+cells+accumulate+in+renal+transplant+patients+and+mediate+CD4(+)+Foxp3(+)+Treg+expansion.">Monocytic myeloid-derived suppressor cells accumulate in renal transplant patients and mediate CD4(+) Foxp3(+) Treg expansion.</a> Am J Transplant. 13 (12), 3123-3131 (2013).</li><li>Tario, J. 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该方案描述了我们在心脏移植的实验鼠模型中用于免疫表达移植物浸润性骨髓细胞亚群的方法，其也适用于不同鼠实验模型中的其他组织。在20psi下的低压细胞分选是分离纯细胞亚群的良好产量的优选方法。保持每个骨髓亚群的纯度对于确定不同骨髓群体之间的抑制能力的确定结果至关重要。然而，其他方法可用于分离各种白细胞群体，例如商业浓缩试剂盒。不管感兴趣的细胞群体，从移植组织?...

该方案描述体外技术，以研究从心脏同种异体移植物分离的组织浸润巨噬细胞的功能，根据其调节T细胞应答的能力。在文献中广泛描述，荧光细胞跟踪染料与流式细胞术结合，是研究特定细胞类型在体外和体内的抑制功能的有力工具。我们的方案遵循用于监测体外淋巴细胞增殖的羧基荧光素琥珀酰亚胺酯（CFSE）方法。 当CFSE标记的细胞分裂时，其后代获得羧基荧光素标记分子数量的一半。通过流式细胞术相应的细胞荧光降低可用于评估细胞分裂，监测抑制性巨噬细胞调节T细胞免疫应答的能力。由于CFSE是基于荧光素的染料，因此它与br相容其他荧光染料范围广，适用于多色流式细胞术。用于表型分析的荧光染料的适当选择对于避免过度的光谱重叠以及不能识别抗体阳性细胞，特别是可见蛋白染料如CFSE 7也是重要的 。 使用荧光染料与测量细胞增殖的替代技术（例如使用放射性标记的胸苷（TdR） 8的胸苷掺入测定法）有很多优点。该测定法利用在有丝分裂细胞分裂期间掺入新染色体DNA链中的氚标记的胸苷（ 3 H-TdR）。与该测定相关的安全性问题是放射性同位素的使用，因为使用闪烁β型计数器来测量从细胞中回收的DNA中的放射性，以便确定细胞分裂的程度。在方法上，氚标记的胸苷屁股ay不足以适应重要的临床实验室限制，如细胞数量少，染色后延迟分析。相反，已经显示CFSE染色可以预防细胞增殖并干扰关键活化标记，如CD69，HLA-DR和CD25 9 。因此，了解各种方法的优点和局限性，特别是在使用多种染料跟踪不同细胞类型的多色研究中，对获得准确和可重现的结果至关重要。

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			<td style="width:123px;">45011282</td>
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			<td height="22" style="height:23px;width:147px;">anti-CD45-APC/Cy7</td>
			<td style="width:111px;">eBioscience</td>
			<td style="width:123px;">47045182</td>
			<td style="width:81px;"></td>
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		<tr height="22">
			<td height="22" style="height:23px;width:147px;">anti-CD4-APC</td>
			<td style="width:111px;">eBioscience</td>
			<td style="width:123px;">17004181</td>
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		<tr height="22">
			<td height="22" style="height:23px;width:147px;">anti-CD8-P/ Cy7</td>
			<td style="width:111px;">eBioscience</td>
			<td style="width:123px;">25008181</td>
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			<td height="22" style="height:23px;width:147px;">LSRII Flow Cytometer</td>
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			<td height="38" style="height:39px;width:147px;">C57BL/6-Foxp3tm1Flv/J&#160;</td>
			<td style="width:111px;">The Jackson Laboratory</td>
			<td style="width:123px;">008374</td>
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			<td height="38" style="height:39px;width:147px;">C57BL/6</td>
			<td style="width:111px;">The Jackson Laboratory</td>
			<td style="width:123px;">000664</td>
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			<td height="38" style="height:39px;width:147px;">Balb/c</td>
			<td style="width:111px;">The Jackson Laboratory</td>
			<td style="width:123px;">000651</td>
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functional characterization of regulatory macrophages that inhibit graft-reactive immunity

移植器官中的巨噬细胞积累早已被认为是同种异体移植排斥的特征1 。免疫原性单核细胞在移植后早期浸润同种异体移植，对移植的器官发生移植反应性反应，并引发器官排斥反应2 。最近的数据表明，抑制性巨噬细胞有助于成功的长期移植3 ，是诱导移植耐受所需要的4 。这表明巨噬细胞个体发育，激活和功能的多维概念，这需要一个新的路线图来分离和分析巨噬细胞功能5 。由于巨噬细胞的可塑性，有必要根据组织环境提供分离和表征巨噬细胞的方法，并根据不同的情况来定义它们的功能。在这里，我们描述作为移植浸润巨噬细胞免疫表征的方案，以及我们用于功能评估其抑制CD8 + T增殖和促进体外 CD4 + Foxp3 + Treg扩增的能力的方法 。

代表性的结果显示了上述方案中描述的门控策略。结果还显示了与移植物浸润巨噬细胞共培养后的T细胞增殖活性的分析。 图4分析了巨噬细胞亚群的体外抑制能力。结果表明，从耐受性受体获得的Ly6C lo Ly6G-巨噬细胞是抑制性的。结果还表明Ly6C int Ly6G +细胞表现出适度的抑制能力。只有Ly6C lo Ly6G -细胞在体外</e...

Watch this Scientific Journal Video about Functional Characterization of Regulatory Macrophages That Inhibit Graft-reactive Immunity at JoVE.com

Functional Characterization of Regulatory Macrophages That Inhibit Graft-reactive Immunity

巨噬细胞是造血系统的塑料细胞，在保护性免疫和体内平衡方面起关键作用。在本报告中，我们描述了优化的体外技术，以表型和功能表征在耐受性条件下积累在移植器官中的移植物浸润调节性巨噬细胞。

抑制接枝反应性免疫的调节性巨噬细胞的功能表征

Macrophage accumulation in transplanted organs has long been recognized as a feature of allograft rejection1. Immunogenic monocytes infiltrate the ...

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从老鼠肠道树突状细胞和巨噬细胞的分离和表征

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and ...

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Forward Genetics Screens Using Macrophages to Identify Toxoplasma gondii Genes Important for Resistance to IFN-&#947;-Dependent Cell Autonomous Immunity

Forward Genetics Screens Using Macrophages to Identify Toxoplasma gondii Genes Important for Resistance to IFN-ÃƒÅ½Ã‚Â³-Dependent Cell Autonomous Immunity

正向遗传学屏风使用巨噬细胞识别<em&gt;弓形虫</em&gt;基因的重要的抗IFN-γ依赖性细胞免疫自主

Toxoplasma gondii, the causative agent of toxoplasmosis, is an obligate intracellular protozoan pathogen. The parasite invades and replicates within ...

Isolation, Characterization and Functional Examination of the Gingival Immune Cell Network

分离，鉴定和牙龈免疫细胞网络的功能性考试

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident ...

Phenotypic and Functional Analysis of Activated Regulatory T Cells Isolated from Chronic Lymphocytic Choriomeningitis Virus-infected Mice

表型和慢性淋巴细胞性脉络丛脑膜炎病毒感染小鼠中分离激活调节性T细胞的功能分析

Regulatory T (Treg) cells, which express Foxp3 as a transcription factor, are subsets of CD4+ T cells. Treg cells play crucial roles in immune tolerance ...

The Isolation, Differentiation, and Quantification of Human Antibody-secreting B Cells from Blood: ELISpot as a Functional Readout of Humoral Immunity

的分离，分化和人类的定量分泌抗体的B细胞从血液：酶联免疫斑点作为体液免疫的功能读出

The hallmark of humoral immunity is to generate functional ASCs, which synthesize and secrete Abs specific to an antigen (Ag), such as a pathogen, and are ...

Phenotypic Characterization of Macrophages from Rat Kidney by Flow Cytometry

通过流式细胞仪大鼠肾脏巨噬细胞的表型特征

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal ...

Using X-ray Crystallography, Biophysics, and Functional Assays to Determine the Mechanisms Governing T-cell Receptor Recognition of Cancer Antigens

利用X射线晶体学，生物物理学，和功能分析，以确定管理机制T细胞癌抗原的受体识别

Human CD8+ cytotoxic T lymphocytes (CTLs) are known to play an important role in tumor control. In order to carry out this function, the cell ...

Isolation, Characterization, and Purification of Macrophages from Tissues Affected by Obesity-related Inflammation

受肥胖相关炎症组织的巨噬细胞的分离、鉴定和纯化

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. ...

Isolation and Characterization of Neutrophil-derived Microparticles for Functional Studies

中性粒细胞衍生微粒的分离与鉴定功能研究

Polymorphonuclear neutrophil-derived microparticles (PMN)-MPs) are lipid bilayer, spherical microvesicles with sizes ranging from 50&#8211;1,000 nm in ...

Flow Cytometric Measurement Of ROS Production In Macrophages In Response To Fc&#947;R Cross-linking

Flow Cytometric Measurement Of ROS Production In Macrophages In Response To FcÃƒÅ½Ã‚Â³R Cross-linking

巨噬细胞 ros 产生的流式细胞仪测量对 fcγr 交联的响应

The oxidative or respiratory burst is used to describe the rapid consumption of oxygen and generation of reactive oxygen species (ROS) by phagocytes in ...