Name: functional characterization of regulatory macrophages that inhibit graft-reactive immunity
Uploaded: 2017-06-07T12:30:00.000+00:00
Duration: 8 min 41 s
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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	<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. R., 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+suppressive+cells+mediate+cardiovascular+transplantation+tolerance+in+mice.">Monocytic suppressive cells mediate cardiovascular transplantation tolerance in mice.</a> J Clin Invest. 120 (7), 2486-2496 (2010).</li><li>Conde, P., 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=DC-SIGN(+)+Macrophages+Control+the+Induction+of+Transplantation+Tolerance.">DC-SIGN(+) Macrophages Control the Induction of Transplantation Tolerance.</a> Immunity. 42 (6), 1143-1158 (2015).</li><li>Ginhoux, F., Schultze, J. L., Murray, P. J., Ochando, J., Biswas, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+insights+into+the+multidimensional+concept+of+macrophage+ontogeny,+activation+and+function.">New insights into the multidimensional concept of macrophage ontogeny, activation and function.</a> Nat Immunol. 17 (1), 34-40 (2016).</li><li>Quah, B. J., Wijesundara, D. K., Ranasinghe, C., Parish, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+fluorescent+target+arrays+for+assessment+of+T+cell+responses+in+vivo.">The use of fluorescent target arrays for assessment of T cell responses in vivo.</a> J Vis Exp. (88), e51627 (2014).</li><li>Tario, J. 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. J., Winn, H. J., Russell, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primarily+vascularized+allografts+of+hearts+in+mice.+The+role+of+H-2D,+H-2K,+and+non-H-2+antigens+in+rejection.">Primarily vascularized allografts of hearts in mice. The role of H-2D, H-2K, and non-H-2 antigens in rejection.</a> Transplantation. 16 (4), 343-350 (1973).</li><li>Liu, F., Kang, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterotopic+heart+transplantation+in+mice.">Heterotopic heart transplantation in mice.</a> J Vis Exp. (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. D., Muirhead, K. A., Pan, D., Munson, M. E., Wallace, P. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracking+immune+cell+proliferation+and+cytotoxic+potential+using+flow+cytometry.">Tracking immune cell proliferation and cytotoxic potential using flow cytometry.</a> Methods Mol Biol. 699, 119-164 (2011).</li></ol>

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，从移植组织获得可行的单细胞悬浮液是最佳表面染色和流式细胞术结果所必需的。不正确的组织操作可能导致细胞损失，因此骨髓亚群的产量低。为了提高产量，请确保处理样品记录冷缓冲液并将细胞保持在具有低细胞粘附性的容器中（聚丙烯管）。在这个方案中，我们使用溶组织溶胶酶A，其被广泛用于许多类型的组织（ 例如肺，心脏，肌肉，骨骼，脂肪，肝，肾，软骨，乳腺，胎盘，血液中的分解）血管，脑和肿瘤）。为了防止细胞在分选时的损失，强烈推荐优化抗体并产生有效的门控策略。在该方案中，我们使用CD11b-Percp / Cy5.5，CD45-APC / eFluor780，Ly6C / APC和Ly6G Pe / Cy7荧光缀合的抗体和流式细胞术来表征鼠巨噬细胞。由于表位Ly6C / G不存在于人体中，因此需要使用CD14 / CD15 / CD16来分选人骨髓细胞15 。 在多色流式细胞术分析中，可能发生选择的荧光染料的可能的光谱重叠。因此，如同befo一样单染色补偿对避免重叠的问题非常有帮助。此外，为了降低背景荧光并获得最佳结果，强烈推荐使用抗体滴定法。减少非特异性结合的另一个重要考虑是使用活力染料。在该协议中，DAPI被用作活力染料。 DAPI（4&#39;，6-二脒基-2-苯基吲哚）是一种与DNA中富含AT的区域强烈结合的蓝色荧光染料。与DNA结合的DAPI的激发最大值为358 nm，发射最大值为461 nm。根据方案使用时，DAPI特异性染色细胞核，具有很少或无细胞质标记，不包括流动分析中的死细胞。然而，根据选择的抗体组，可以使用其它活性染料，例如7-氨基生物素霉素D（7-AAD）和碘化丙啶（PI）。准确的死细胞排除和活细胞鉴定是成功监测体外 T细胞的先决条件，因为它是重要的重要的是具有能够以最小的细胞活力和功能破坏的淋巴细胞增殖的程序。 如上所述，用细胞跟踪染料如CFSE进行细胞标记和增殖分析是关键的步骤。作为文献中描述的一个例子，必须小心排除死亡/死亡细胞，以便在使用CFSE 16时获得均匀分布和可区分的子峰。根据染色面板，有许多市售的细胞追踪染料可供选择。作为细胞跟踪染料的替代品，进行胸苷滴定测定以评估癌症研究中淋巴细胞和其他细胞的增殖13,14 。胸苷掺入方案通过放射性标记的3H-或14C-胸苷的测量来评估细胞分裂期间DNA的复制。虽然这个方法我相当敏感和成熟，胸苷滴定技术的主要缺点是它涉及放射性，不能在单细胞水平提供信息。另一方面，CFSE流式细胞术分析提供了有关淋巴细胞亚群反应的清晰信息，并且具有较少的内部和内部变异性。

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。

<table><tbody><tr><td>RPMI 1640 Media</td><td>Life Technologies</td><td>11875119</td><td></td></tr><tr><td>10% FBS</td><td>Life Technologies</td><td>26140-079</td><td></td></tr><tr><td>1,000x 2-mercaptoethanol</td><td>Life Technologies</td><td>21985023</td><td></td></tr><tr><td>100x Pen/Strep</td><td>Life Technologies</td><td>15140122</td><td></td></tr><tr><td>100x L-glutamine</td><td>Life Technologies</td><td>25030081</td><td></td></tr><tr><td>100x Non Esential amino acids</td><td>Corning</td><td>25025039</td><td></td></tr><tr><td>1 M HEPES buffer&amp;nbsp;</td><td>Corning</td><td>25060CL</td><td></td></tr><tr><td>100 mM Sodium Pyruvate</td><td>ThermoScientific</td><td>SH30239.01</td><td></td></tr><tr><td>Penicilin/Streptavidyn</td><td>ThermoScientific</td><td>15140122</td><td></td></tr><tr><td>Collagenese A</td><td>Roche</td><td>70381322</td><td></td></tr><tr><td>DPBS (w/o calcium&amp;amp;magnesium)</td><td>Corning</td><td>21031CV</td><td></td></tr><tr><td>70 &amp;micro;m Cell strainer</td><td>Fisher</td><td>22363548</td><td></td></tr><tr><td>ACK lysis buffer</td><td>Life Technologies</td><td>A10492-01</td><td></td></tr><tr><td>DAPI</td><td>Sigma</td><td>32670-5mg-F</td><td></td></tr><tr><td>CFSE-FITC</td><td>Invitrogen</td><td>C34554</td><td></td></tr><tr><td>Dynabeads Mouse T cell activator CD3/CD28</td><td>Life Technologies</td><td>11452D</td><td></td></tr><tr><td>96 well&amp;nbsp; U-bottom plate</td><td>Corning</td><td>353077</td><td></td></tr><tr><td>Antibodies:</td><td></td><td></td><td></td></tr><tr><td>anti-Ly6C-APC</td><td>eBioscience</td><td>175932-80</td><td></td></tr><tr><td>anti-Ly6G-Pe/Cy7</td><td>Biolegend</td><td>127617</td><td></td></tr><tr><td>anti-CD11b-Percp/Cy5.5</td><td>eBioscience</td><td>45011282</td><td></td></tr><tr><td>anti-CD45-APC/Cy7</td><td>eBioscience</td><td>47045182</td><td></td></tr><tr><td>anti-CD4-APC</td><td>eBioscience</td><td>17004181</td><td></td></tr><tr><td>anti-CD8-P/ Cy7</td><td>eBioscience</td><td>25008181</td><td></td></tr><tr><td>LSRII Flow Cytometer</td><td>BD Bioscience</td><td></td><td></td></tr><tr><td>FACSDiva Software</td><td>BD Bioscience</td><td></td><td></td></tr><tr><td>C57BL/6-Foxp3tm1Flv/J&amp;nbsp;</td><td>The Jackson Laboratory</td><td>008374</td><td></td></tr><tr><td>C57BL/6</td><td>The Jackson Laboratory</td><td>000664</td><td></td></tr><tr><td>Balb/c</td><td>The Jackson Laboratory</td><td>000651</td><td></td></tr></tbody></table>

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 -细胞在体外促进CD4 + Foxp3 + Treg 的扩增 。在一起，数据支持结论，移植浸润CD11b + Ly6C lo Ly6G -巨噬细胞具有报告与单核细胞衍生的抑制细胞12相关的许多性质的结论包括其抑制CD8 T细胞增殖的能力13并促进CD4 + Foxp3 + Treg扩增14 。 <img alt="图1" src="/files/ftp_upload/54242/54242fig1.jpg" /> 图1:动物模型。 （ A ）如前所述，将Balb / c心（H2-d）移植到完全同种异体C57BL / 6（H2-b）中。显示接受者的气管静脉和供体的肺动脉和升主动脉的腹主动脉的吻合。如我们最近报道的那样，在第0,2和4天，用250μg抗CD40L mAb（克隆MR1）处理接受者小鼠的耐受诱导。通过腹部触诊每隔一天监测移植功能。拒绝被定义为完全停止可触觉的心跳，并通过直接可视化确认<html>剖腹手术。 （ B ）代表性图像和（ C ）LN和脾脏（彩色为红色）和同种异体移植物（紫色）的解剖位置说明<a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54242/54242fig1large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图2" src="/files/ftp_upload/54242/54242fig2.jpg" /> 图2:巨噬细胞分选策略。从左上角开始，白细胞首先通过大小门控，然后将单体细胞与碎片和团块区分开来。从单体中，死细胞被排除在DAPI阴性部分上。来自活细胞，CD45 + CD11b +用于鉴定骨髓细胞。基于Ly6C和Ly6G表达进一步鉴定三个骨髓细胞群体。="_ blank"&gt;请点击此处查看此图的较大版本。 <img alt="图3" src="/files/ftp_upload/54242/54242fig3.jpg" /> 图3:淋巴细胞分选策略。淋巴细胞分选策略的代表性流式细胞术结果。从左上方，淋巴细胞群体的门根据前向和侧向散射。排除碎屑和团块。从单体中，死细胞被排除在DAPI阴性部分上。从活细胞中，（ A ）CD8 + CFSE +双阳性细胞和（ B ）含有一部分Foxp3 +阳性细胞的所有CD4 + T细胞被门控。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54242/54242fig3large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图4" src="/files/ftp_upload/54242/54242fig4.jpg"/&gt; 图4:抑制试验。 （ A ）每个骨髓亚群的体外抑制能力。 CD8 + T细胞增殖通过CFSE稀释法监测与骨髓亚群共培养96小时后。 （ B ）每个骨髓亚群的体外 Treg扩增。流式细胞术分析Foxp3在与骨髓亚型共培养96小时后在CD4 + T细胞上的表达。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54242/54242fig4large.jpg" target="_blank">请点击此处查看此图的较大版本。</a>

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

Nanyang Technological University

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 Protocol of Mouse Monocyte-derived Dendritic Cells and Their Subsequent In Vitro Activation with Tumor Immune Complexes

Dendritic cells (DC) are heterogeneous cell populations that differ in their cell membrane markers, migration patterns and distribution, and in their antigen presentation and T cell activation capacities. Since most vaccinations of experimental tumor models require millions of DC, they are widely isolated from the bone marrow or spleen. However, these DC significantly differ from blood and tumor DC in their responses to immune complexes (IC), and presumably to other Syk-coupled lectin receptors. Importantly, given the sensitivity of DC to danger-associated molecules, the presence of endotoxins or antibodies that crosslink activation receptors in one of the isolating steps could result in the priming of DC and thus affect the parameters, or at least the dosage, required to activate them. Therefore, here we describe a detailed protocol for isolating MoDC from blood and tumors while avoiding their premature activation. In addition, a protocol is provided for MoDC activation with tumor IC, and their subsequent analyses.

Isolation Protocol of Mouse Monocyte-derived Dendritic Cells and Their Subsequent In Vitro Activation with Tumor Immune Complexes

Monocyte-derived DC (MoDC) can sense minor amounts of danger-associated molecules and are therefore easily primed. We provide a detailed protocol for the isolation of MoDC from blood and tumors and their activation with immune complexes while highlighting key precautions that should be considered in order to avoid their premature activation.

小鼠单核细胞衍生树突状细胞的分离及与肿瘤免疫配合物的后体外活化

Dendritic cells (DC) are heterogeneous cell populations that differ in their cell membrane markers, migration patterns and distribution, and in their ...

科研

癌症研究

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 ...

免疫与感染

Depletion and Reconstitution of Macrophages in Mice

Macrophages are critical players in the innate immune response to infectious challenge or injury, initiating the innate immune response and directing the acquired immune response. Macrophage dysfunction can lead to an inability to mount an appropriate immune response and as such, has been implicated in many disease processes, including inflammatory bowel diseases. Macrophages display polarized phenotypes that are broadly divided into two categories. Classically activated macrophages, activated by stimulation with IFN&gamma; or LPS, play an essential role in response to bacterial challenge whereas alternatively activated macrophages, activated by IL-4 or IL-13, participate in debris scavenging and tissue remodeling and have been implicated in the resolution phase of inflammation. During an inflammatory response in vivo, macrophages are found amid a complex mixture of infiltrating immune cells and may participate by exacerbating or resolving inflammation. To define the role of macrophages in situ in a whole animal model, it is necessary to examine the effect of depleting macrophages from the complex environment. To ask questions about the role of macrophage phenotype in situ, phenotypically defined polarized macrophages can be derived ex vivo, from bone marrow aspirates and added back to mice, with or without prior depletion of macrophages. In the protocol presented here clodronate-containing liposomes, versus PBS injected controls, were used to deplete colonic macrophages during dextran sodium sulfate (DSS)-induced colitis in mice. In addition, polarized macrophages were derived ex vivo and transferred to mice by intravenous injection. A caveat to this approach is that clodronate-containing liposomes deplete all professional phagocytes, including both dendritic cells and macrophages so to ensure the effect observed by depletion is macrophage-specific, reconstitution of phenotype by adoptive transfer of macrophages is necessary. Systemic macrophage depletion in mice can also be achieved by backcrossing mice onto a CD11b-DTR background, which is an excellent complementary approach. The advantage of clodronate-containing liposome-mediated depletion is that it does not require the time and expense involved in backcrossing mice and it can be used in mice regardless of the background of the mice (C57BL/6, BALB/c, or mixed background).

Macrophages play a central role in homeostasis and pathology in many tissues. The protocol presented here describes methods for depleting macrophages in vivo, deriving polarized macrophages from bone marrow aspirates, and adoptively transferring macrophages into mice. These techniques allow determination of the role that polarized macrophages play in health and disease.

在小鼠体内的巨噬细胞的枯竭和重建

Macrophages are critical players in the innate  immune response to infectious challenge or injury, initiating the innate immune  response and directing ...

Studying the Effects of Tumor-Secreted Paracrine Ligands on Macrophage Activation using Co-Culture with Permeable Membrane Supports

Tumor-derived paracrine signaling is an overlooked component of local immunosuppression and can lead to a permissive environment for continued cancer growth and metastasis. Paracrine signals can involve cell-cell contact between different cell types, such as PD-L1 expressed on the surface of tumors interacting directly with PD-1 on the surface of T cells, or the secretion of ligands by a tumor cell to affect an immune cell. Here we describe a co-culture method to interrogate the effects of tumor-secreted ligands on immune cell (macrophage) activation. This straightforward procedure utilizes commercially available 0.4 &#181;m polycarbonate membrane permeable supports and standard tissue culture plates. In the process described, macrophages are cultured in the upper chamber and tumor cells in the lower chamber. The presence of the 0.4 &#181;m barrier allows for the study of intercellular signaling without the confounding variable of physical contact, because the two cell types share the same medium and exposure to paracrine ligands. This approach can be combined with others, such as genetic alterations of the macrophage (e.g., isolation from genetic knock-out mice) or tumor (e.g., CRISPR-mediated alterations) to study the role of specific secreted factors and receptors. The approach also lends itself to standard molecular biological analyses such as quantitative reverse transcription polymerase chain reaction (qRT-PCR) or Western blot analysis, without the need for flow sorting to separate the two cell populations. Enzyme-linked immunosorbent assays (ELISAs) can similarly be utilized to measure secreted ligands to better understand the dynamic interaction of cell signaling in the multiple cell type context. Duration of co-culture can also be varied for the study of temporally regulated events. This co-culture method is a robust tool that facilitates the study of tumor-secreted signals in the immune context.

Here, we present a method using permeable membrane supports to facilitate the study of non-contact paracrine signaling used by tumor cells to suppress the immune response. The system is amenable to studying the role of tumor-secreted factors in dampening macrophage activation.

研究肿瘤分泌性副体结扎对宏噬细胞活化的影响，使用可渗透膜支架共培养

Tumor-derived paracrine signaling is an overlooked component of local immunosuppression and can lead to a permissive environment for continued cancer ...

Isolation of Murine Peritoneal Macrophages to Carry Out Gene Expression Analysis Upon Toll-like Receptors Stimulation

During infection and inflammation, circulating monocytes leave the bloodstream and migrate into tissues, where they differentiate into macrophages. Macrophages express surface Toll-like receptors (TLRs), which recognize molecular patterns conserved through evolution in a wide range of microorganisms. TLRs play a central role in macrophage activation which is usually associated with gene expression alteration. Macrophages are critical in many diseases and have emerged as attractive targets for therapy. In the following protocol, we describe a procedure to isolate murine peritoneal macrophages using Brewer&#8217;s thioglycollate medium. The latter will boost monocyte migration into the peritoneum, accordingly this will raise macrophage yield by 10-fold. Several studies have been carried out using bone marrow, spleen or peritoneal derived macrophages. However, peritoneal macrophages were shown to be more mature upon isolation and are more stable in their functionality and phenotype. Thus, macrophages isolated from murine peritoneal cavity present an important cell population that can serve in different immunological and metabolic studies. Once isolated, macrophages were stimulated with different TLR ligands and consequently&#160;gene expression was evaluated.

We describe here a simple protocol to isolate murine peritoneal macrophages. This procedure is followed by RNA extraction to carry out gene expression analysis upon Toll-like receptors stimulation.

小鼠腹腔巨噬细胞的分离，以贯彻基因表达分析后Toll样受体刺激

During infection and inflammation, circulating monocytes leave the bloodstream and migrate into tissues, where they differentiate into macrophages. ...

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 ...

Mouse Model of Alloimmune-induced Vascular Rejection and Transplant Arteriosclerosis

Vascular rejection that leads to transplant arteriosclerosis (TA) is the leading representation of chronic heart transplant failure. In TA, the immune system of the recipient causes damage of the arterial wall and dysfunction of endothelial cells and smooth muscle cells. This triggers a pathological repair response that is characterized by intimal thickening and luminal occlusion. Understanding the mechanisms by which the immune system causes vasculature rejection and TA may inform the development of novel ways to manage graft failure. Here, we describe a mouse aortic interposition model that can be used to study the pathogenic mechanisms of vascular rejection and TA. The model involves grafting of an aortic segment from a donor animal into an allogeneic recipient. Rejection of the artery segment involves alloimmune reactions and results in arterial changes that resemble vascular rejection. The basic technical approach we describe can be used with different mouse strains and targeted interventions to answer specific questions related to vascular rejection and TA.

We describe a protocol for aortic interposition grafting in mice. The goal of the protocol is to provide a model with which to study pathological processes and therapeutic strategies relevant to alloimmune reactions in arteries and the resultant arterial changes that contribute to organ transplant failure.

的同种免疫引起的血管性排斥反应和移植物动脉硬化小鼠模型

Vascular rejection that leads to transplant arteriosclerosis (TA) is the leading representation of chronic heart transplant failure. In TA, the immune ...

医学

In Vitro and In Vivo Assessment of T, B and Myeloid Cells Suppressive Activity and Humoral Responses from Transplant Recipients

The main concern in transplantation is to achieve specific tolerance through induction of regulatory cells. The understanding of tolerance mechanisms requires reliable models. Here, we describe models of tolerance to cardiac allograft in rat, induced by blockade of costimulation signals or by upregulation of immunoregulatory molecules through gene transfer. Each of these models allowed in vivo generation of regulatory cells such as regulatory T cells (Tregs), regulatory B cells (Bregs) or regulatory myeloid cells (RegMCs). In this manuscript, we describe two complementary protocols that have been used to identify and define in vitro and in vivo regulatory cell activity to determine their responsibility in tolerance induction and maintenance. First, an in vitro suppressive assay allowed rapid identification of cells with suppressive capacity on effector immune responses in a dose dependent manner, and can be used for further analysis such as cytokine measurement or cytotoxicity. Second, the adoptive transfer of cells from a tolerant treated recipient to a newly irradiated grafted recipient, highlighted the tolerogenic properties of these cells in controlling graft directed immune responses and/or converting new regulatory cells (termed infectious tolerance). These methods are not restricted to cells with known phenotypic markers and can be extended to any cell population. Furthermore, donor directed allospecificity of regulatory cells (an important goal in the field) can be assessed by using third party donor cells or graft either in vitro or in vivo. Finally, to determine the specific tolerogenic capacity of these regulatory cells, we provide protocols to assess the humoral anti-donor antibody responses and the capacity of the recipient to develop humoral responses against new or former known antigens. The models of tolerance described can be used to further characterize regulatory cells, to identify new biomarkers, and immunoregulatory molecules, and are adaptable to other transplantation models or autoimmune diseases in rodent or human.

In Vitro and In Vivo Assessment of T, B and Myeloid Cells Suppressive Activity and Humoral Responses from Transplant Recipients

Here, we present a protocol to induce tolerance in transplantation, and assess in vitro and in vivo the suppressive capacity of distinct cell subsets from the recipient and the immune status of the recipient toward donor or exogenous antigens.

体外和活体评估移植受体的 T、B 和髓细胞抑制活性和体液反应

The main concern in transplantation is to achieve specific tolerance through induction of regulatory cells. The understanding of tolerance mechanisms ...

Assessment of Antibody-based Drugs Effects on Murine Bone Marrow and Peritoneal Macrophage Activation

Macrophages are phagocytic innate immune cells, which initiate immune responses to pathogens and contribute to healing and tissue restitution. Macrophages are equally important in turning off inflammatory responses. We have shown that macrophages stimulated with intravenous immunoglobulin (IVIg) can produce high amounts of the anti-inflammatory cytokine, interleukin 10 (IL-10), and low levels of pro-inflammatory cytokines in response to bacterial lipopolysaccharides (LPS). IVIg is a polyvalent antibody, primarily immunoglobulin Gs (IgGs), pooled from the plasma of more than 1,000 blood donors. It is used to supplement antibodies in patients with immune deficiencies or to suppress immune responses in patients with autoimmune or inflammatory conditions. Infliximab, a therapeutic anti-tumor necrosis factor alpha (TNF&#945;) antibody, has also been shown to activate macrophages to produce IL-10 in response to inflammatory stimuli. IVIg and other antibody-based biologics can be tested to determine their effects on macrophage activation. This paper describes methods for derivation, stimulation, and assessment of murine bone marrow macrophages activated by antibodies in vitro and murine peritoneal macrophages activated with antibodies in vivo. Finally, we demonstrate the use of western blotting to determine the contribution of specific cell signaling pathways to anti-inflammatory macrophage activity. These protocols can be used with genetically modified mice, to determine the effect of a specific protein(s) on anti-inflammatory macrophage activation. These techniques can also be used to assess whether specific biologics may act by changing macrophages to an IL-10-producing anti-inflammatory activation state that reduces inflammatory responses in vivo. This can provide information on the role of macrophage activation in the efficacy of biologics during disease models in mice, and provide insight into a potential new mechanism of action in people. Conversely, this may caution against the use of specific antibody-based biologics to treat infectious disease, particularly if macrophages play an important role in host defense against that infection.

Antibody-based drugs have revolutionized treatment for inflammatory diseases. In addition to having direct effects on specific targets, antibodies can activate macrophages to become anti-inflammatory. This protocol describes how anti-inflammatory macrophage activation can be assessed in vitro, using mouse bone marrow macrophages, and in vivo, using peritoneal macrophages.

抗体药物对小鼠骨髓及腹腔巨噬细胞活化作用的评价

Macrophages are phagocytic innate immune cells, which initiate immune responses to pathogens and contribute to healing and tissue restitution. Macrophages ...

Mesenchymal Stem Cell Regulation of Macrophage Phagocytosis; Quantitation and Imaging

Mesenchymal stem cells (MSC) have traditionally been studied for their regenerative properties, but more recently, their immunoregulatory characteristics have been at the forefront. They interact with and regulate immune cell activity. The focus of this study is the MSC regulation of macrophage phagocytic activity. Macrophage (M&#934;) phagocytosis is an important part of the innate immune system response to infection, and the mechanisms through which MSC modulate this response are under active investigation. Presented here is a method to study M&#934; phagocytosis of non-opsonized zymosan particles conjugated to a pH-sensitive fluorescent molecule while in co-culture with MSC. As phagocytic activity increases and the labeled zymosan particles are enclosed within the acidic environment of the phagolysosome, the fluorescence intensity of the pH-sensitive molecule increases. With the appropriate excitation and emission wavelengths, phagocytic activity is measured using a fluorescent spectrophotometer and kinetic data is presented as changes in relative fluorescent units over a 70 min period. To support this quantitative data, the change in the phagocytic activity is visualized using dynamic imaging. Results using this method demonstrate that when in co-culture, MSC enhance M&#934; phagocytosis of non-opsonized zymosan of both naive and IFN-&#947; treated M&#934;. These data add to the current knowledge of MSC regulation of the innate immune system. This method can be applied in future investigations to fully delineate the underlying cellular and molecular mechanisms.

Presented here is a protocol to quantify and produce dynamic images of mesenchymal stem cell (MSC) mediated regulation of macrophage (M&#934;) phagocytosis of non-opsonized yeast (zymosan) particles that are conjugated to a pH-sensitive fluorescent molecule.

间充质干细胞调节巨噬细胞吞噬作用;定量和成像

Mesenchymal stem cells (MSC) have traditionally been studied for their regenerative properties, but more recently, their immunoregulatory characteristics ...