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培养基巯基乙醇。 </li></ol> 2.同种异体移植物隔离和单细胞悬浮液注意:Corry和合作者最初描述了肺动脉和下腔静脉的移植和吻合技术，并可在Liu＆Kangs ="xref"&gt; 10,11 （ 图1A ）。 <ol><li>在感应室中用4-5％异氟烷麻醉移植的小鼠。通过颈椎脱位牺牲。 </li><li>用标准剪刀做中线腹部切口，取出腹部内容物使主动脉局部化。 注意:移植的心脏将在受体腹主动脉右侧（ 图1B ）。 </li><li>使用精细的尖牙镊子轻轻拉出移植物，并将其立即放入冰冷的RPMI培养基中。 注意:使用手术剪刀将移植物与主动脉分离可能会损伤移植物，并导致一些组织保持粘附于受体。 </li><li>在收集所有移植物后，将其移至无菌培养罩中进行组织处理。将移植物置于陪替氏培养皿中，使用无菌，钝性，显微外科剪刀将组织切成小块（1毫米）。 </li><li>用锋利的牙齿forceps，将片段转移到具有5mL胶原酶A（0.1mg / mL无菌1x PBS中的胶原酶）的50mL管中。在37℃浴中孵育1小时。 </li><li>加入5 mL RPMI培养基中和胶原酶，并借助1 mL注射器的柱塞将样品转移通过100μm过滤器。 </li><li>在4℃下将样品以400xg的速度旋转5分钟。 </li><li>将沉淀重悬于1mL的ACK裂解缓冲液中。混合均匀并在4℃下孵育5分钟。 </li><li>加入1mL RPMI培养基以中和裂解缓冲液，并在4℃下以400×g的速度旋转样品5m。 </li><li>将细胞沉淀重悬于200μLRPMI培养基中，并将其转移至5 mL聚丙烯圆底管中。 注意:聚丙烯管支持较少的粘附。此外，将电池维持在低温（例如4℃）下降低粘附性。 </li></ol> 3.使用荧光激活细胞分选分离移植物浸润性巨噬细胞ING <ol><li>通过使用Fc受体阻断单克隆抗体（大鼠抗小鼠CD16 / 32）阻断骨髓细胞上的不需要的特异性结合。在表面染色前15分钟加入每个样品1-2μL。 </li><li>用抗小鼠CD11b Percp / Cy5.5（RPMI培养基中的最终浓度为0.6μg/μL），抗小鼠CD45 APC / eFluor780（0.6μg/μL），抗小鼠Ly6C APC（2μg/μL）和抗小鼠Ly6G Pe / Cy7（2μg/μL）。用铝箔覆盖管，以保护荧光抗体免受光照，并将冰箱中的管在4℃孵育45分钟。 注意:对于单染色补偿，准备阴性对照管（无染色）和管，每个荧光染料单独标记细胞。为了帮助建立门，特异性对照可以特别用于Ly6C（IgG2a）和Ly6G（IgG2b）。 </li><li>用RPMI培养基洗涤细胞两次，并在4℃下以400xg旋转5分钟。使用台盼蓝和血细胞计数细胞并将其在每1×10 6个细胞中的1mL RPMI培养基中稀释。分选前，将样品转移到5 mL，70μm的细胞过滤器管帽上。加入DAPI（1μg/ mL）作为细胞活力标记，并进行分类。 </li><li>因为巨噬细胞是敏感和脆弱的细胞，将排序条件设置为20 psi，并使用100μm喷嘴大小，以使用4通纯度模式分离巨噬细胞。 </li><li>在5 mL聚丙烯管中，用1 mL RPMI培养基准备收集管。 </li><li>使用分拣机软件打开新的实验，并用空白面板选择空白实验来定义设置。 </li><li> 设置一个点图，显示所有白细胞的正向（FSC）对（ vs. ）侧向散射（SSC）和门，不包括具有最低前向和侧向散射的碎片和团块。从这个父门，创建一个新的点图显示SSC 与 DAPI和门DAPI单元格。 <ol><li>在这个新门槛的人口中，创建广告显示CD11b 对 CD45和门对双阳性（CD11b + CD45 + ）巨噬细胞和嗜中性粒细胞的图。 </li><li>从这里，创建一个显示Ly6C 与 Ly6G的最终点图，并筛选所需的群体:Ly6C hi Ly6G - ，Ly6C lo Ly6G -和Ly6C int Ly6G + （ 图2 ）。 </li></ol></li><li>分选后，检查纯度和细胞活力（&gt; 90％），并在4℃下以400xg旋转收集管5分钟。使用台盼蓝和血细胞计数器计数细胞，并在完整的RPMI培养基中以所需浓度（ 例如， 1×10 6巨噬细胞/ mL）重悬。 </li><li>在96孔圆底板中每孔50×10 3个巨噬细胞，终体积为100μL的完整RPMI培养基。将细胞在37℃和5％CO 2下不受干扰至少24小时。 </li></ol>隔离n的T细胞使用荧光激活细胞分选注意:C57BL / 6-Foxp3tm1Flv / Jn是用单克隆红色荧光蛋白（mRFP）共同标记表达Foxp3（叉头盒P3）基因的细胞的X连锁靶向敲入小鼠株。 <ol><li>麻醉并牺牲C57BL / 6和C57BL / 6-Foxp3tm1Flv / J（H-2b）小鼠，如先前在步骤2.1中所述。分离脾脏和淋巴结（LN:腹股沟，腰椎，轴，臂和颈），并迅速将其置于冰冷的RPMI培养基中。 </li><li>为了隔离LN，将鼠标放置在仰卧位置，从底部到顶部进行中线皮肤切口，小心地切开腹膜并轻轻地将皮肤分开。一旦收集所有腹股沟，腰椎，腋窝，肱动脉和子宫颈LN（ 图1C ，红色着色），切开腹膜并分离肠道左上方的脾脏。 </li><li>使用放置的100μm滤器分解脾脏和LN在50mL管的顶部。使用1-mL注射器的柱塞，轻轻按压组织。用RPMI培养基冲洗过滤器多次，直到清洁干净。 注意:每只小鼠的LN和脾脏可以在同一根管中汇集在一起​​。 </li><li>在4℃下以400xg的速度旋转5分钟。 </li><li>将沉淀重悬于2 mL ACK裂解缓冲液中。混合均匀并在4℃下孵育5分钟。 </li><li>加入2mL RPMI培养基以中和裂解缓冲液。在4℃下以400xg的速度旋转5分钟。 </li><li>将细胞沉淀重悬于200μLRPMI培养基中，并将其转移至5 mL聚苯乙烯圆底管中。 </li><li>用于抗小鼠CD4 APC（0.6μg/μL）和/或抗小鼠CD8 PeCy7（2μg/μL）的染色。用铝箔覆盖管，以保护荧光抗体免受光照，并将管在冰箱中孵育4℃45分钟。 注意:对于单染色补偿，准备阴性对照管（无染色）和细胞与每个荧光染料单独标记的细胞管。 </li><li>用RPMI培养基洗涤细胞两次，并在4℃下以400xg旋转5分钟。使用台盼蓝和血细胞计数器计数细胞。 注:CFSE染料以羧基荧光素二乙酸酯琥珀酰亚胺酯粉末的形式提供，通常为50μg。加入18μLDMSO至一瓶CFSE，最终储存液为5 mM，储存于-20°C几个月。 </li><li>对于CFSE标记，在PBS中以浓度高达10 6个细胞/ mL的浓度，从储备溶液中以5μMCFSE的最终工作浓度重新悬浮CD8染色的细胞。如前所述，将它们在20℃的浴中孵育5分钟。 注意:（关键步骤）对于低浓度（≤106/ mL）的细胞，必须在添加的蛋白质存在下对细胞进行标记以缓冲CFSE的毒性作用。因此，PBS可以含有5％FBS。请注意，如果我使用ium，游离氨基酸可能通过竞争CFSE降低标签效率。 </li><li>用2 mL RPMI培养基中和CFSE。在4℃下以400xg旋转5分钟。每1×10 6 CD8 T细胞添加1 mL RPMI培养基。 </li><li>分选前，将样品转移到5 mL管帽上的70μm细胞过滤器上。加入50μL1x DAPI（1μg/ mL）作为细胞活力标记，并进行分选。 注意:1x表示试剂与其他成分的比例为1:1。 </li><li>将排序条件设置为20 psi和100μm喷嘴大小，以隔离双阳性CD8 + CFSE + T细胞（ 图3A ）。 </li><li>使用1 mL RPMI培养基在5 mL聚丙烯管中准备收集管，并进行分类。 </li><li>使用分拣机软件打开新的实验，并用空白面板选择空白实验来定义设置。 </li><li> 对于CD4 + T细胞分离，设置一个点图播放FSC对SSC和淋巴细胞上的门，不包括碎片以及大颗粒细胞，如树突状细胞。 <ol><li>从这个父门，创建一个新的点图显示SSC与DAPI和门DAPI单元格。 </li><li>在这个新门控的人群中，创建一个点图，显示SSC与CD4分离所有CD4 +细胞（ 图3B ）。或者，可以使用抗CD3单克隆抗体来确保纯T细胞群的分离。 注意:小部分CD4 + T细胞（5-10％）应该是Foxp3 + mRFP + 。 </li></ol></li><li>分选后，检查纯度和细胞活力（&gt; 90％），并在4℃下以400xg旋转收集管5分钟。使用台盼蓝和血细胞计数器计数细胞。使用每2×10 6个 T细胞1mL的完全RPMI培养基重悬。 </li><li>通过用预先分选的巨噬细胞培养T细胞来建立抑制测定。板10 x 10 4 CD4 + T和10×10 4个 CD8 + CFSE + T细胞在最终体积为100μL的完整RPMI培养基中。在37℃和5％CO 2孵育96小时。 注意:以1:4的比例使用巨噬细胞和T细胞。 </li><li>通过在使用它们之前将T细胞激活剂CD3 / CD28磁珠洗涤在2mL PBS中来刺激T细胞分裂，以清除含叠氮化物的缓冲液。每孔加入5μL小鼠T细胞激活剂CD3 / CD28珠。 注意:磁珠与抗原呈递细胞大小相似，并与抗CD3和抗CD28单克隆抗体偶联，为T细胞的活化和扩增提供了一种简单的方法。 </li></ol>

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作者没有什么可以披露的。

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 height="22" style="height:23px;width:147px;">anti-CD45-APC/Cy7</td>
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			<td style="width:123px;">47045182</td>
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			<td height="22" style="height:23px;width:147px;">anti-CD4-APC</td>
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			<td style="width:123px;">17004181</td>
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			<td height="22" style="height:23px;width:147px;">anti-CD8-P/ Cy7</td>
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			<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>
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			<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 ...

functional-characterization-regulatory-macrophages-that-inhibit-graft

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

8.2K 次观看.  Icahn School of Medicine at Mount Sinai. 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。 

视频: Functional Characterization of Regulatory Macrophages That Inhibit Graft-reactive Immunity

Isolation and Characterization of Dendritic Cells and Macrophages from the Mouse Intestine

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and food antigens while concomitantly remaining poised to mount inflammatory responses toward invasive pathogens1,2. Antigen presenting cells, particularly DCs and macrophages, play critical roles in maintaining intestinal immune homeostasis via their ability to sense and appropriately respond to the microbiota3-14. Efficient isolation of intestinal DCs and macrophages is a critical step in characterizing the phenotype and function of these cells. While many effective methods of isolating intestinal immune cells, including DCs and macrophages, have been described6,10,15-24, many rely upon long digestions times that may negatively influence cell surface antigen expression, cell viability, and/or cell yield. Here, we detail a methodology for the rapid isolation of large numbers of viable, intestinal DCs and macrophages. Phenotypic characterization of intestinal DCs and macrophages is carried out by directly staining isolated intestinal cells with specific fluorescence-labeled monoclonal antibodies for multi-color flow cytometric analysis. Furthermore, highly pure DC and macrophage populations are isolated for functional studies utilizing CD11c and CD11b magnetic-activated cell sorting beads followed by cell sorting.

Here, we detail a methodology for the rapid isolation of mouse intestinal dendritic cells (DCs) and macrophages. Phenotypic characterization of intestinal DCs and macrophages is performed using multi-color flow cytometric analysis while magnetic bead enrichment followed by cell sorting is used to yield highly pure populations for functional studies.

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 ...

科研

免疫与感染

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

Toxoplasma gondii, the causative agent of toxoplasmosis, is an obligate intracellular protozoan pathogen. The parasite invades and replicates within virtually any warm blooded vertebrate cell type. During parasite invasion of a host cell, the parasite creates a parasitophorous vacuole (PV) that originates from the host cell membrane independent of phagocytosis within which the parasite replicates. While IFN-dependent-innate and cell mediated immunity is important for eventual control of infection, innate immune cells, including neutrophils, monocytes and dendritic cells, can also serve as vehicles for systemic dissemination of the parasite early in infection. An approach is described that utilizes the host innate immune response, in this case macrophages, in a forward genetic screen to identify parasite mutants with a fitness defect in infected macrophages following activation but normal invasion and replication in na&#239;ve macrophages. Thus, the screen isolates parasite mutants that have a specific defect in their ability to resist the effects of macrophage activation. The paper describes two broad phenotypes of mutant parasites following activation of infected macrophages: parasite stasis versus parasite degradation, often in amorphous vacuoles. The parasite mutants are then analyzed to identify the responsible parasite genes specifically important for resistance to induced mediators of cell autonomous immunity. The paper presents a general approach for the forward genetics screen that, in theory, can be modified to target parasite genes important for resistance to specific antimicrobial mediators. It also describes an approach to evaluate the specific macrophage antimicrobial mediators to which the parasite mutant is susceptible. Activation of infected macrophages can also promote parasite differentiation from the tachyzoite to bradyzoite stage that maintains chronic infection. Therefore, methodology is presented to evaluate the importance of the identified parasite gene to establishment of chronic infection.

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

Forward genetics is a powerful approach to identify genes in intracellular pathogens important for resistance to cell autonomous immunity. The current approach uses innate immune cells, specifically macrophages, to identify novel Toxoplasma gondii genes important for immune evasion.

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

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident immune cell populations in the oral mucosa and gingiva. We have established a technique for the isolation and study of immune cells from murine gingival tissues, an area of constant microbial exposure and a vulnerable site to a common inflammatory disease, periodontitis. Our protocol allows for a detailed phenotypic characterization of the immune cell populations resident in the gingiva, even at steady state. Our procedure also yields sufficient cells with high viability for use in functional studies, such as the assessment of cytokine secretion ex vivo. This combination of phenotypic and functional characterization of the gingival immune cell network should aid towards investigating the mechanisms involved in oral immunity and periodontal homeostasis, but will also advance our understanding of the mechanisms involved in local immunopathology.

We have established a technique for the isolation, phenotypic characterization and functional analysis of immune cells from murine gingiva.

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

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 and homeostasis maintenance by regulating the immune response. The primary role of Treg cells is to suppress the proliferation of effector T (Teff) cells and the production of cytokines such as IFN-&#947;, TNF-&#945;, and IL-2. It has been demonstrated that Treg cells&#39; ability to inhibit the function of Teff cells is enhanced during persistent pathogen infection and cancer development. To clarify the function of Treg cells under resting or inflamed conditions, a variety of in vitro suppression assays using mouse or human Treg cells have been devised. The main aim of this study is to develop a method to compare the differences in phenotype and suppressive function between resting and activated Treg cells. To isolate activated Treg cells, mice were infected with lymphocytic choriomeningitis virus (LCMV) clone 13 (CL13), a chronic strain of LCMV. Treg cells isolated from the spleen of LCMV CL13-infected mice exhibited both the activated phenotype and enhanced suppressive activity compared with resting Treg cells isolated from na&#239;ve mice. Here, we describe the basic protocol for ex vivo phenotype analysis to distinguish activated Treg cells from resting Treg cells. Furthermore, we describe a protocol for the measurement of the suppressive activity of fully activated Treg cells.

Here, we describe a protocol to analyze the phenotype of regulatory T (Treg) cells isolated from na&#239;ve and chronic lymphocytic choriomeningitis virus-infected mice. In addition, we provide a process to evaluate the suppressive activity of the Treg cells.

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

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 used for host defense. For the quantitative determination of the functional status of the humoral immune response of an individual, both serum Abs and circulating ASCs are commonly measured as functional readouts. In humans, peripheral blood is the most convenient and readily accessible sample that can be used for the determination of the humoral immune response elicited by host B cells. Distinct B-cell subsets, including ASCs, can be isolated directly from peripheral blood via selection with lineage-specific Ab-conjugated microbeads or via cell sorting with flow cytometry. Moreover, purified na&#239;ve and memory B cells can be activated and differentiated into ASCs in culture. The functional activities of ASCs to contribute to Ab secretion can be quantified by ELISpot, which is an assay that converges enzyme-linked immunoabsorbance assay (ELISA) and western blotting technologies to enable the enumeration of individual ASCs at the single-cell level. In practice, the ELISpot assay has been increasingly used to evaluate vaccine efficacy because of the ease of handling of a large number of blood samples. The methods of isolating human B cells from peripheral blood, the differentiation of B cells into ASCs in vitro, and the employment of ELISpot for the quantification of total IgM- and IgG-ASCs will be described here.

Human peripheral blood is commonly used for the assessment of the humoral immune response. Here, the methods for isolating human B cells from peripheral blood, differentiating human B cells into antibody (Ab)-secreting B cells (ASCs) in culture, and enumerating the total IgM- and IgG-ASCs via an ELISpot assay are described.

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

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal disease. Macrophages are heterogeneous cells that have been implicated in kidney injury. Macrophages may be classified into two different phenotypes: classically activated macrophages (M1 macrophages), that release pro-inflammatory cytokines and promote fibrosis; and alternatively activated macrophages (M2 macrophages) that are associated with immunoregulatory and tissue-remodeling functions. These macrophage phenotypes need to be discriminated and analyzed to determine their contribution to renal injury. However, there are scarce studies reporting consistent phenotypic and functional information about macrophage subtypes in inflammatory renal disease models, especially in rats. This fact may be related to the limited macrophage markers used in rats, contrary to mice. Therefore, novel strategies are necessary to quantify and characterize the renal content of these infiltrating cells in a reliable way. This manuscript details a protocol for kidney digestion and further phenotypic and quantitative analysis of macrophages from rat kidneys by flow cytometry. Briefly, kidneys were incubated with collagenase and total macrophages were identified according to the dual presence of CD45 (leukocytes common antigen) and CD68 (PAN macrophage marker) in live cells.This was followed by surface staining of CD86 (M1 marker) and CD163 (M2 marker). Rat peritoneal macrophages were used as positive control for macrophage marker detection by flow cytometry. Our protocol resulted in low cellular mortality and allowed characterization of different intracellular and surface protein markers, thus limiting the loss of cellular integrity observed in other protocols. Moreover, this procedure allows the use of macrophages for further techniques, including cell sorting and mRNA or protein expression studies, among others.

This manuscript describes a detailed protocol for phenotypic and quantitative analysis of resident macrophages from rat kidneys by flow cytometry. The resulting stained cells can be also used for other applications, including cell sorting, gene expression analysis or functional studies, thus increasing the information obtained in the experimental model.

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

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 surface-expressed T-cell receptor (TCR) must functionally recognize human leukocyte antigen (HLA)-restricted tumor-derived peptides (pHLA). However, we and others have shown that most TCRs bind sub-optimally to tumor antigens. Uncovering the molecular mechanisms that define this poor recognition could aid in the development of new targeted therapies that circumnavigate these shortcomings. Indeed, present therapies that lack this molecular understanding have not been universally effective. Here, we describe methods that we commonly employ in the laboratory to determine how the nature of the interaction between TCRs and pHLA governs T-cell functionality. These methods include the generation of soluble TCRs and pHLA and the use of these reagents for X-ray crystallography, biophysical analysis, and antigen-specific T-cell staining with pHLA multimers. Using these approaches and guided by structural analysis, it is possible to modify the interaction between TCRs and pHLA and to then test how these modifications impact T-cell antigen recognition. These findings have already helped to clarify the mechanism of T-cell recognition of a number of cancer antigens and could direct the development of altered peptides and modified TCRs for new cancer therapies.

Here, we describe methods that we commonly employ in the laboratory to determine how the nature of the interaction between the T-cell receptor and tumor antigens, presented by human leukocyte antigens, governs T-cell functionality; these methods include protein production, X-ray crystallography, biophysics, and functional T-cell experiments.

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

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. Diet-induced obesity (DIO) is a valuable model in studying the role of macrophage heterogeneity; however, adequate macrophage isolations are difficult to acquire from inflamed tissues. In this protocol, we outline the isolation steps and necessary troubleshooting guidelines derived from our studies for obtaining a suitable population of tissue-resident macrophages from mice following 18 weeks of high-fat (HFD) or high-fat/high-cholesterol (HFHCD) diet intervention. This protocol focuses on three hallmark tissues studied in obesity and atherosclerosis including the liver, white adipose tissues (WAT), and the aorta. We highlight how dualistic usage of flow cytometry can achieve a new dimension of isolation and characterization of tissue-resident macrophages. A fundamental section of this protocol addresses the intricacies underlying tissue-specific enzymatic digestions and macrophage isolation, and subsequent cell-surface antibody staining for flow cytometric analysis. This protocol addresses existing complexities underlying fluorescent-activated cell sorting (FACS) and presents clarifications to these complexities so as to obtain broad range characterization from adequately sorted cell populations. Alternate enrichment methods are included for sorting cells, such as the dense liver, allowing for flexibility and time management when working with FACS. In brief, this protocol aids the researcher to evaluate macrophage heterogeneity from a multitude of inflamed tissues in a given study and provides insightful troubleshooting tips that have been successful for favorable cellular isolation and characterization of immune cells in DIO-mediated inflammation.

This protocol allows a researcher to isolate and characterize tissue-resident macrophages in various hallmark inflamed tissues extracted from diet-induced models of metabolic disorders.

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

Polymorphonuclear neutrophil-derived microparticles (PMN)-MPs) are lipid bilayer, spherical microvesicles with sizes ranging from 50&#8211;1,000 nm in diameter. MPs are a newly evolving, important part of cell-to-cell communication and signaling machinery. Because of their size and the nature of their release, until recently MP existence was overlooked. However, with improved technology and analytical methods their function in health and disease is now emerging. The protocols presented here are aimed at isolating and characterizing PMN-MPs by flow cytometry and immunoblotting. Moreover, several implementation examples are given. These protocols for MP isolation are fast, low-cost, and do not require the use of expensive kits. Furthermore, they allow for the labeling of MPs following isolation, as well as pre-labeling of source cells prior to MP release, using a membrane-specific fluorescent dye for visualization and analysis by flow cytometry. These methods, however, have several limitations including purity of PMNs and MPs and the need for sophisticated analytical instrumentation. A high-end flow cytometer is needed to reliably analyze MPs and minimize false positive reads due to noise or auto-fluorescence. The described protocols can be used to isolate and define MP biogenesis, and characterize their markers and variation in composition under different stimulating conditions. Size heterogeneity can be exploited to investigate whether the content of membrane particles versus exosomes is different, and whether they fulfill different roles in tissue homeostasis. Finally, following isolation and characterization of MPs, their function in cellular responses and various disease models (including, PMN-associated inflammatory disorders, such as Inflammatory Bowel Diseases or Acute Lung Injury) can be explored.

New protocols are described here to isolate and characterize microparticles derived from human and mouse neutrophils. These protocols utilize ultracentrifugation, flow cytometry, and immunoblotting techniques to analyze microparticle content, and they can be used to study the role of microparticles derived from various cell types in cellular function.

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

The oxidative or respiratory burst is used to describe the rapid consumption of oxygen and generation of reactive oxygen species (ROS) by phagocytes in response to various immune stimuli. ROS generated during immune activation exerts potent antimicrobial activity primarily through the ability of ROS to damage DNA and proteins, causing death of microorganisms. Being able to measure ROS production reproducibly and with ease is necessary in order to assess the contribution of various pathways and molecules to this mechanism of host defense. In this paper, we demonstrate the use of fluorescent probes and flow cytometry to detect ROS production. Although widely used, fluorescent measurement of ROS is notoriously problematic, especially with regards to measurement of ROS induced by specific and not mitogenic stimuli. We present a detailed methodology to detect ROS generated as a result of specific Fc&#947;R stimulation beginning with macrophage generation, priming, staining, Fc&#947;R cross-linking, and ending with flow cytometric analysis.

This study demonstrates the use of flow cytometry to detect reactive oxygen species (ROS) production resulting from activation of the Fc&#947;R. This method can be used to assess changes in the antimicrobial and redox signaling function of phagocytes in response to immune complexes, opsonized microorganisms, or direct Fc&#947;R 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 ...