Name: use of a monocyte monolayer assay to evaluate fcγ receptor-mediated phagocytosis
Uploaded: 2017-01-02T13:00:00.000+00:00
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Description: Watch this Scientific Journal Video about Use of a Monocyte Monolayer Assay to Evaluate FcÎ³ Receptor-mediated Phagocytosis at JoVE.com

The authors thank the Canadian Blood Services for a Graduate Fellowship Program Award to T.N.T. This research received financial support from the Canadian Blood Services' Centre for Innovation, funded by the federal government (Health Canada) and the provincial and territorial ministries of health. The views herein do not reflect the views of the federal, provincial, or territorial governments in Canada.

获得批准后，由研究伦理委员会/机构审查委员会使用人的样本，以及从人类所有的献血者获得签字同意。 MMA的，也可以以类似的方式作为人类试验用小鼠的PBMC进行的，下面的动物使用伦理批准。在MMA利用无菌组织培养技术。 注:参见图1。 注:虽然我们在试验过程中推荐使用2级生物控制柜保持无菌技术，因为该方法只是一个"短期"培养法，确实没有足够的时间进行细菌污染检测。因此，如果一个2级生物控制柜不存在，而不是购买一只是为了该测定，该测定可以一个生物防护柜外进行，一个开放的长凳上。使用37℃培养箱，用5％ 的 CO 2，然而，这不是一个选项，必须使用最佳结果。 1.外周血单个核细胞分离<ol><li>来自健康供体或通过使用含有酸 - 柠檬酸盐 - 葡萄糖（ACD）抗凝剂（黄 - 顶管）真空采血管静脉穿刺的患者获得人全血。 注:全血可在室温（18-22℃）达36小时继续下一个步骤11之前被存储在ACD。通常，1-2的全血10毫升的vacutainer管是足以用于测定。 </li><li>稀释全血1:1体积/体积在温暖的完全RPMI培养基（RPMI-1640添加有10％胎牛血清，20mM的HEPES，和0.01毫克/毫升庆大霉素）。 </li><li>隔离从稀释的全血用密度梯度离心如制造商推荐的外周血单核细胞（PBMC），（见材料清单）。层的稀释的血液非常缓慢的密度梯度（升温至室温，18-22℃）。 注:最小化Mixi的量通过以逐滴的方式小心地分层血液混合物或通过使用移液管在用于血液的最佳分离界面纳克。 <ol><li>允许血液混合物缓慢通过将吸管尖接近密度梯度和通过使血液混合物运行非常缓慢向下管侧的密度梯度的顶部层上。 </li><li>根据实验的规模，层10毫升3毫升浓度梯度的顶部血液混合物（在15毫升管）或层35毫升15毫升的密度梯度（的顶端血液混合物在50mL的管）。通常情况下，10毫升的全血得到千万的PBMC，用一些供体到供体的变化。 注意:这是非常重要的是，不存在与密度梯度的血液混合物的混合。血混合物应层上密度梯度慢慢上升，直到所有的血液是在密度梯度顶部。 </li><li>离心分层混合物在700 XG 30分钟没有刹车。该CENTRifuged混合物应分离成5层（从顶部到底部）:血浆，血沉棕黄层（含有PBMCs）中，密度梯度材料，粒细胞和红细胞。 </li><li>卸下并丢弃大部分等离子体，并仔细检索血沉棕黄层（PBMC）内容转换成使用巴斯德吸管和吸入灯泡一个新的15毫升管中。 注:除去血沉棕黄层的有效通过在巴斯德吸管施加吸力，同时执行围绕层的外侧的圆周运动，以对管的吸移管的尖端进行。 </li></ol></li><li>通过在洗涤之间，在350 xg离心离心10分钟（全制动器）洗涤所述分离的PBMC中的pH 7.3磷酸盐缓冲的盐水（PBS）的三倍。在复溶完全RPMI培养基PBMC沉淀。根据粒料的大小，3-7毫升介质就足够了。 </li><li>采用台盼蓝和血球计数PBMC。只计算不被台盼蓝染色的那些细胞。 ReconstitutE中的外周血单个核细胞，以1,750,000个细胞/ mL完全RPMI培养基。 </li><li>种子400微升（700000细胞）成8室滑动的每个孔中。孵育所述滑动在37℃，饱和湿度的组织培养培养箱中（补充有5％的CO 2）1小时，以使单核细胞/巨噬细胞附着。 </li></ol>单核细胞的粘2.前处理注:此步骤只是必要的，如果寻找各种方法来抑制或增强吞噬作用。 <ol><li>预治疗粘附的单核细胞与感兴趣的任何药物（S）或化合物（S）。重构该药物（S）或其他测试材料使用完全RPMI培养基中的所需浓度。 注意:例如，免疫球蛋白的200微克/毫升，通常使用使用人单核细胞时，得到的吞噬作用的95-100％抑制。 </li><li>吸液和丢弃含有从8室滑动任何非粘附细胞的1小时温育（步骤1.6之后）之后的上清液。用400取代它药物或其他治疗的微升孵育在37℃下1小时。 <ol><li>当吸并在8室幻灯片替代解决方案，一定要控制流体的流动，使弱粘附细胞不升空。此外，在一个时间只有2-3井空工作，避免干燥井。 注意:通常情况下，每一个处理是在技术一式三份进行。 </li></ol></li></ol> 3.调理作用的R 2 R 2红血细胞注意:此处所用的，R 2，R 2是从血液采集中心（加拿大血液服务）中获得，但它们也可商购。调理，R 2，R 2红细胞被用作的FcγR介导的吞噬作用的阳性对照。幼稚，R 2，R 2应在4℃下储存在阿氏液（以延长保质期）长达1个月。阿氏的解决方案是在公司内部制作和0.8组成％w / v的柠檬酸三钠（二水合物），1.9％w / v的葡萄糖，0.42％w / v的氯化钠，以及0.05％w / v的柠檬酸（一水合物）。当R 2，R 2细胞是不可用，其他的Rh-表型的细胞，如R 1 R 2，R 1，R 1，R 1 R，或R 2 R，都可以使用。 <ol><li>洗涤R 2 R 2（CDE / CDE）的红血细胞在PBS中，总的以350 xg离心使用离心三次，每次5分钟。 注意:需要的RBC的量取决于实验的大小和能重新计算。常洗红细胞过量，因为红细胞各自洗涤由于裂解或除去上清液中期间丢失。例如，在具有5的治疗和2的控制，所有的技术一式三份进行的实验，有一个总的21孔中。 21井用400微升/孔的1.25％RBC的混合物表明，填充105微升，调理需要红细胞。 200红细胞微升应先洗涤和150＆＃181:L应调理，以确保有红细胞的吞噬作用步骤的足够量。 </li><li> Opsonize的洗涤，R 2，R 2丸粒用1:1体积/体积多克隆抗D抗体从人血清，并在37℃下孵育1小时，用间歇混合。 注意:如果多克隆抗D抗体不可用，也可以使用单克隆抗D抗体，其是市售的，或抗D是通常用于Rh免疫预防，这可以从血库或输血而获得服务。优化测试应该在开始时设置了测定，每当切换大量未滴定多克隆抗体或切换到的单克隆抗体来进行，。这是确定的抗人球蛋白试验的3+和4+与人均100单核细胞计数70-90之间吞噬红细胞的吞噬作用的结果（IAT）结果所需的最佳浓度或抗D的量。 </li><li>洗opsonizeðR 2 R 2使用离心350 XG的每5分钟的PBS共三次。 注:R 2 R 2的成功调理可以通过执行间接IAT确认。简言之，二次多克隆抗人抗体被添加到绑定红细胞表面主opsonizing抗体，并且可以在血细胞凝集的形式观察到的放大的信号。详细的制造商协议可以在补充材料的文件中找到。 </li><li>重新组建洗R 2 R 2球团至1.25％V / V使用完全RPMI网上平台。 注:过量调理，R 2，R 2可以存储在阿氏液在4℃下长达一周。 </li></ol> 4. Fc受体介导的吞噬作用<ol><li>从8室滑动吸出药物或介质上清液，并添加1.25％体积/体积，R 2，R 2混合物的400微升。孵育在37℃下2小时。 </li><li>前前后后呃孵育2小时，取出使用制造商的适配器室。轻拍掉过量的R 2，R 2在纸巾上。确保滑不干燥。 </li><li>填充100-mL烧杯中，用PBS。淹没和缓慢移动的来回滑动（30-40杆），以去除大部分的未吞噬R 2 R 2洗的幻灯片。 </li><li>取下PBS的幻灯片。轻拍多余的PBS用纸巾或组织和空气干燥的幻灯片。 </li><li>固定在100％甲醇中风干滑板45秒。然后，风干固定滑动。 注:幻灯片可以用另一种方法，是为下游染色更兼容，如Grünwald的-Giemsa染色21或赖特-吉姆萨染色22是固定的。 </li><li>使用内部制造的ELVANOL安装介质（或其他市售的安装介质），并添加盖玻片安装的幻灯片。 注:ELVANOL安装介质由15％w / v的婆lyvinyl醇树脂和30％体积/体积甘油的PBS中。将混合物加热直到所有的树脂溶解和甘油是均匀混合。这可以与其它市售的安装平台代替。 </li><li>允许安装到量化之前干通宵。 </li></ol> 5.吞噬的量化<ol><li>使用相位差显微镜和40X物镜，通过手动计数至少200个单核细胞和吞噬R 2 R 2的这些单核细胞中的数字量化吞噬活动的量。在每个手一个计数器来同时量化的单核细胞的数量和吞噬R 2 R 2的数目。 </li><li>通过单核细胞的数量除以吞噬R 2 R 2的数目，并通过100 Express中的数据为平均值（平均吞噬指数）±平均值（SEM）的标准误差相乘获得平均吞噬指数。 </li&gt; </ol>

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The authors have nothing to disclose.

该MMA是一个艰苦的技术，它需要在这两个组织培养和显微镜的专业知识。有几个关键的步骤，以确保成功:1）代单核细胞单层的; 2）红细胞的调理作用，和3）手册定量。单核细胞单层不很强烈粘附于腔滑动，因此，生理pH必须在整个测定11保持和PBMC中的足够数量应接种。剧烈吸移，这可能会干扰粘附细胞，应避免使用。一种方法是总是删除并从腔室的同一角落添加解决方案，并保证了动作是缓慢而稳定的。同样地，在最后的洗涤步骤，以除去过量的红细胞，运动应该是缓慢而稳定的。这可确保干扰最小的单分子层，同时还除去大部分未吞噬红细胞。洗涤不充分会导致污染红细胞高的背景下，使得手动QUAntification困难。其次，R 2，R 2红细胞必须充分调理，以获得为80-120吞噬控制的平均吞噬指数。这个希望的吞噬范围撞击容易数量之间的平衡（ 例如，超过5单核细胞的吞噬红细胞难以准确量化）和维持吞噬的统计分析足够量。调理作用的程度可以通过一个IAT被确认，并且需要对4+ 3+之间的读数。的R 2，R 2红细胞应当有在洗涤过程中过量的裂解，当上清液变成暗红色，或者被丢弃时在吞噬一个显著减少实验的观察，由于在存储单元的老化。最后，使用显微镜定量手册可能会非常棘手，比较实验室人员之间和实验之间计数时尤其如此。通过检查每个同场好，或者干脆计数更多细胞，可以得到更一致的计数。建议侧并排培训，经验丰富的技师，并使用指定的一套培训幻灯片中。 在MMA的一个主要批评是手动量化步骤的主观性。然而，有足够的训练，可以在不同的柜台获得一致性。另一个限制是单核细胞吞噬能力，在R 2，R 2表面抗原表达水平，这是数据的变化与人体标本打交道时，源固有的捐助者对捐助者的差异。 其他替代技术可用于检查的FcγR介导的吞噬作用。大多数商业试剂盒的利用荧光输出来监控吞噬（ 例如，生物粒子，pH敏感的荧光蛋白，或IgG标记的荧光胶乳珠）。使用荧光输出并提供更多的客观量化，但有也需要精读代尔的可用性，成本和与使用荧光显微镜或流式细胞仪，以及在市售试剂盒随后依赖相关的培训。 最后，该测定可根据所研究的问题进行修改。例如，在测试的吞噬作用的药物抑制时，单核细胞可以预先处理或用药物和调理红细胞（ 即，竞争测定）两者共同培养。不同的亚型，嵌合抗体，或重组构建体抗体的下游信号传导，也可以进行测试。在一个普遍的抗原空血液24开发最近的突破，MMA的可与各种抗体这些抗原空红细胞初始画面被利用来评估是否有确实在引发吞噬作用降低的功效。

The monocyte monolayer assay (MMA) is an in vitro assay originally developed to better predict blood transfusion outcomes in patients with auto- or alloantibodies to red blood cells (RBCs)1-5. By assessing the effect of anti-RBC antibodies in mediating Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis using this in vitro assay, it is possible to predict the clinical outcome in vivo. Indeed, the MMA has been used successfully to avoid immune destruction of antibody-bound RBCs, despite the transfusion of serologically incompatible blood5. The typical pre-transfusion procedure for compatibility testing, also termed crossmatching, involves serological methods that include typing the patient's blood for ABO and Rh antigens and screening for the presence of anti-RBC antibodies in the patient6. Blood matched for ABO/Rh is selected, and if antibodies are present, an attempt to identify them is made so that blood for transfusion can be further selected to avoid these antigens. An ideal crossmatch result occurs when all donor blood is serologically compatible with the patient's blood, which reduces the risk of post-transfusion hemolysis7. However, this system falls short for the small group of patients who have become alloimmunized upon repeated transfusion or pregnancy. These patients produce alloantibodies against specific RBC antigens. Some produce antibodies to antigens of very high frequency in the general population, and thus become progressively more difficult to crossmatch8,9. Adding to the complexity, not all alloantibodies are clinically significant; in other words, the binding of an alloantibody to RBCs detected by a serology test does not necessarily result in hemolysis when antigen-positive, incompatible blood is transfused. The MMA was originally developed to assess the potential clinical significance of serologically incompatible blood in a transfusion setting1-5.
Since extravascular hemolysis of antibody-bound RBCs is known to be mediated by the mononuclear phagocyte system, primary monocytes/macrophages are utilized in the development of diagnostic assays. The first assay to study the interaction of monocytes, RBCs, and antibodies was published in 1975, but the sub-optimal conditions used led only to rosette formation (the binding of RBCs to the periphery of the monocyte), and no phagocytosis was observed10. Significant modifications to the assay were made by several groups, leading to an assay for which the level of phagocytosis of alloantibody-bound RBCs could be correlated to the clinical outcome of hemolysis1-5. Recently, the optimal storage conditions of clinical samples and further optimization of assay conditions were examined to enhance the utility of a clinical MMA crossmatch using autologous patient samples11.
Three other diagnostic techniques have been employed in addition to the MMA in predicting transfusion outcomes: the 51Cr release test, the rosette test, and the chemiluminescence test (CLT). In the 51Cr release test, the patient is injected with 51Cr-labeled donor RBCs, and the half-life of the labeled RBCs is monitored and is predictive of post-transfusion survival or clearance12,13. As this method uses radioactive materials, it is rarely performed anymore. The rosette test involves mixing and incubating monocytes with RBCs and quantifying the level of rosette formation (with no phagocytosis)14. The clinical significance of antibodies in vivo involves active phagocytosis by macrophages found in the spleen and/or the liver; thus, this method does not provide a relevant readout of phagocytosis. The CLT uses luminol to monitor the oxidative burst during monocyte phagocytosis of RBC, since luminol fluoresces blue when oxidized in the phagosome15. This method is good, but contamination by neutrophils can confound the readout. Parallel comparisons have been made to evaluate the sensitivity, practicality, and reproducibility of the four available methods, and both the CLT and MMA were ranked superior16. However, the CLT has been mainly utilized in assessing hemolytic disease of the fetus and newborn (HDFN), and the assay's optimal pH of 8.0 might compromise the level of phagocytosis11.
In addition to its diagnostic and clinical utility, the MMA has been modified for other research purposes. Indeed, the MMA can not only serve as a functional assay to address discrepancies between serology and biology, it has also been used to retrospectively investigate the cause of hemolysis after intravenous immunoglobulin (IVIG) therapy17. It has also been used to examine the structure-function of chemical inhibitors of Fc&#947;R-mediated phagocytosis18-20 and to study the downstream signaling of Fc&#947;R-mediated phagocytosis21. In our laboratory, in addition to using a human MMA, we are developing a murine MMA using primary mouse peripheral blood mononuclear cells (PBMCs) and autologous RBCs. The rationale is to screen antibodies that can induce Fc&#947;R-mediated phagocytosis as an intermediate to developing an in vivo autoimmune hemolytic anemia (AIHA) mouse model (unpublished data). The various modifications focus on different aspects of the IgG antibody and Fc&#947;R interaction that induce phagocytosis.

<table><tbody><tr><td>Acid citrate dextrose (ACD) vacutainers</td><td>BD</td><td>REF364606</td><td></td></tr><tr><td>RPMI 1640</td><td>Sigma</td><td>R8758</td><td></td></tr><tr><td>HEPES</td><td>Bioshop</td><td>HEP003.100</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Multicell&amp;nbsp;</td><td>080150</td><td></td></tr><tr><td>Gentamicin</td><td>Gibco</td><td>15710-64</td><td></td></tr><tr><td>Ficoll-Paque PLUS</td><td>GE</td><td>17-1440-03</td><td>https://www.gelifesciences.com/</td></tr><tr><td>Phosphate buffered saline</td><td>Sigma</td><td>D8537</td><td></td></tr><tr><td>8-chamber slides</td><td>Lab-Tek-ll</td><td>154534</td><td></td></tr><tr><td>R&lt;sub&gt;2&lt;/sub&gt;R&lt;sub&gt;2&lt;/sub&gt; (cDE/cDE) red blood cells</td><td>Canadian Blood Services</td><td></td><td>Commercially available (&lt;em&gt;e.g.&lt;/em&gt;, http://www.bio-rad.com/en-ca/product/reagent-red-blood-cells)</td></tr><tr><td>Polyclonal anti-D from human serum</td><td>Gamma Biologics</td><td>DIN 02247724</td><td>Can be substituted with commercially available monoclonal anti-D or with Rh immune globulin</td></tr><tr><td>100% methanol</td><td>Caledon</td><td>6700-1-42</td><td></td></tr><tr><td>Polyvinyl alcohol resin</td><td>Sigma</td><td>P8136</td><td>Can be substituted with commercially available mount</td></tr><tr><td>UltraPure glycerine</td><td>Invitrogen</td><td>15514-011</td><td></td></tr><tr><td>Cover slips</td><td>VWR</td><td>48366 067</td><td></td></tr><tr><td>Novaclone anti-IgG</td><td>Immucorgamma</td><td>5461023</td><td>Optional for IAT (http://www.fda.gov/downloads/biologicsbloodvaccines/.../ucm081743.pdf)</td></tr></tbody></table>

use of a monocyte monolayer assay to evaluate fc&#947; receptor-mediated phagocytosis

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly versatile in vitro assay that can be modified to examine different aspects of antibody and Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis in both research and clinical settings. The assay utilizes adherent monocytes from peripheral blood mononuclear cells isolated from mammalian whole blood. MMA has been described for use in both human and murine investigations. These monocytes express Fc&#947;Rs (e.g., Fc&#947;RI, Fc&#947;RIIA, Fc&#947;RIIB, and Fc&#947;RIIIA) that are involved in immune responses. The MMA exploits the mechanism of Fc&#947;R-mediated interactions, phagocytosis in particular, where antibody-sensitized red blood cells (RBCs) adhere to and/or activate Fc&#947;Rs and are subsequently phagocytosed by the monocytes. In vivo, primarily tissue macrophages found in the spleen and liver carry out Fc&#947;R-mediated phagocytosis of antibody-opsonized RBCs, causing extravascular hemolysis. By evaluating the level of phagocytosis using the MMA, different aspects of the in vivo Fc&#947;R-mediated process can be investigated. Some applications of the MMA include predicting the clinical relevance of allo- or autoantibodies in a transfusion setting, assessing candidate drugs that promote or inhibit phagocytosis, and combining the assay with fluorescent microscopy or traditional Western immunoblotting to investigate the downstream signaling effects of Fc&#947;R-engaging drugs or antibodies. Some limitations include the laboriousness of this technique, which takes a full day from start to finish, and the requirement of research ethics approval in order to work with mammalian blood. However, with diligence and adequate training, the MMA results can be obtained within a 24-h turnover time.

按照图1和上面概述的过程的关键步骤，MMA的可再现地进行。 IVIG用作吞噬的图2中的抑制的例子。 IVIG已知结合并阻断Fc受体，从而抑制吞噬作用的下游结果。通过滴定IVIG的使用量，以剂量依赖的抑制观察到，其中浓度大于200微克/毫升导致接近100％的抑制和浓度低于0.5微克/毫升在所有（ 图2A）不抑制。当吞噬指数被变换和规范化到R 2，R 2阳性对照作为0％抑制，用3微克的IC 50 / mL的可被确定（ 图2B）的抑制曲线。 除了持续进行测定，quantifica该测定的灰有时可能是困难的。 图3是各种MMA幻灯片相差显微镜图像的集合。用显微镜的经验，人们可以区分从污染红细胞（ 图3D），或从吞噬RBC（ 图3B）液泡的单核细胞。应的量化（ 图3E和F）过程中应避免细胞和/或碎屑的致密簇。此外，应避免过度opsonizing的R 2 R 2的RBC，这将导致提高的吞噬该overcrowds单核细胞内部和准确的定量（ 图3C）干扰。 <img alt="图1" src="/files/ftp_upload/55039/55039fig1.jpg" /> 图1的MMA的示意图。一个一步一步描绘在MMA的关键步骤:从全血中分离PBMC，喂养坚持monocytES在加入调理的，R 2，R 2，和洗涤室载玻片。 <a href="http://ecsource.jove.com/files/ftp_upload/55039/55039fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/55039/55039fig2.jpg" /> 图2.静脉的IgG（IVIG） 抑制使用MMA 体外吞噬。 IVIG是已知的抑制吞噬作用并用作在人的MMA的抑制控制。相比于未处理的，R 2，R 2控制时（A）中的IVIG滴定导致吞噬的剂量依赖性抑制。结果显示了平均值±平均值的n = 3的实验（SEM）的标准误差。统计分析使用学生t检验进行:**P≤0.01和***P≤0.001。 （B）用的IC 50°的IVIG抑制曲线F 3微克/毫升（虚线）。结果表明归一化到未处理的R 2 R 2（0％抑制）的数据;平均n = 3的实验±SEM。 <a href="http://ecsource.jove.com/files/ftp_upload/55039/55039fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/55039/55039fig3.jpg" /> 下40X放大率样品载玻片的图3.相衬显微镜图像。 （ 一 ）完善的幻灯片，其中单核细胞平均（带有鲜明的光环发光黑色箭头）吞噬1-2个R 2 R 2，除了极少数污染，未吞噬R 2 R 2的背景（白色箭头）。 （ 二 ）在本幻灯片中所示，单核细胞有时也扩大空泡（白色箭头），它可以被误认为是吞噬R 2 R 2。 （C &lt;/强&gt;）当R 2，R 2已经超过调理，超过4-5吞噬，R 2，R 2每单核细胞（黑色箭头）呈现准确计数困难，由于R 2，R 2是在单核细胞和不同的细胞内挤边界可以不被区分。 （D）的幻灯片洗涤不充分导致了丰富的R 2 R 2污染（白色箭头），这可能会被误认为是坚持红细胞。 （E）有时候，单核细胞和红细胞污染可能形成集群。簇也表明细菌污染，应该避免的。 （F）的单核细胞可能会形成大的聚集体，这应该量化期间应避免。使用字段随机选择查看，面板A是当MMA已经被正确执行是通常观察到的。比例尺是20微米。 <a href="http://ecsource.jove.com/files/ftp_upload/55039/55039fig3large.jpg" target="_blank">请点击此处</a>查看该图的放大版本。

Watch this Scientific Journal Video about Use of a Monocyte Monolayer Assay to Evaluate FcÎ³ Receptor-mediated Phagocytosis at JoVE.com

Use of a Monocyte Monolayer Assay to Evaluate Fc&#947; Receptor-mediated Phagocytosis

The monocyte monolayer assay (MMA) is an in vitro assay that utilizes isolated primary monocytes obtained from mammalian peripheral whole blood to evaluate Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis.

单核细胞单层试验中用于评估Fcγ受体介导的吞噬作用

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly ...

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Universidad San Sebastian

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JoVE Journal

Immunology and Infection

17.9K 次观看.  University of Toronto. The monocyte monolayer assay (MMA) is an in vitro assay that utilizes isolated primary monocytes obtained from mammalian peripheral whole blood to evaluate Fcγ receptor (FcγR)-mediated phagocytosis.

视频: Use of a Monocyte Monolayer Assay to Evaluate Fcγ Receptor-mediated Phagocytosis

Determining the Phagocytic Activity of Clinical Antibody Samples

Antibody-driven phagocytosis is induced via the engagement of Fc receptors on professional phagocytes, and can contribute to both clearance as well as pathology of disease. While the properties of the variable domains of antibodies have long been considered critical to in vivo function, the ability of antibodies to recruit innate immune cells via their Fc domains has become increasingly appreciated as a major factor in their efficacy, both in the setting of recombinant monoclonal antibody therapy, as well as in the course of natural infection or vaccination1-3.
Importantly, despite its nomenclature as a constant domain, the antibody Fc domain does not have constant function, and is strongly modulated by IgG subclass (IgG1-4) and glycosylation at Asparagine 2974-6. Thus, this method to study functional differences of antigen-specific antibodies in clinical samples will facilitate correlation of the phagocytic potential of antibodies to disease state, susceptibility to infection, progression, or clinical outcome.
Furthermore, this effector function is particularly important in light of the documented ability of antibodies to enhance infection by providing pathogens access into host cells via Fc receptor-driven phagocytosis7. Additionally, there is some evidence that phagocytic uptake of immune complexes can impact the Th1/Th2 polarization of the immune response8.
Here, we describe an assay designed to detect differences in antibody-induced phagocytosis, which may be caused by differential IgG subclass, glycan structure at Asn297, as well as the ability to form immune complexes of antigen-specific antibodies in a high-throughput fashion. To this end, 1 &mu;m fluorescent beads are coated with antigen, then incubated with clinical antibody samples, generating fluorescent antigen specific immune complexes. These antibody-opsonized beads are then incubated with a monocytic cell line expressing multiple Fc&gamma;Rs, including both inhibitory and activating. Assay output can include phagocytic activity, cytokine secretion, and patterns of Fc&gamma;Rs usage, and are determined in a standardized manner, making this a highly useful system for parsing differences in this antibody-dependent effector function in both infection and vaccine-mediated protection9.

We present a high-throughput flow cytometric assay to determine the phagocytic activity of antigen-specific antibodies from clinical samples, utilizing fluorescent antigen-coated beads and a monocytic cell line expressing multiple Fc receptors&#x2014;providing receptor usage and phagocytic activity determinations in a standardized and reproducible fashion for any antigen of interest.

确定临床抗体样品的吞噬活性

Antibody-driven phagocytosis is induced via the engagement of Fc receptors on professional phagocytes, and can contribute to both clearance as well as ...

科研

免疫与感染

High Throughput Fluorometric Technique for Assessment of Macrophage Phagocytosis and Actin Polymerization

The goal of fluorometric analysis is to serve as an efficient, cost effective, high throughput method of analyzing phagocytosis and other cellular processes. This technique can be used on a variety of cell types, both adherent and non-adherent, to examine a variety of cellular properties. When studying phagocytosis, fluorometric technique utilizes phagocytic cell types such as macrophages, and fluorescently labeled opsonized particles whose fluorescence can be extinguished in the presence of trypan blue. Following plating of adherent macrophages in 96-well plates, fluorescent particles (green or red) are administered and cells are allowed to phagocytose for varied amounts of time. Following internalization of fluorescent particles, cells are washed with trypan blue, which facilitates extinction of fluorescent signal from bacteria which are not internalized, or are merely adhering to the cell surface. Following the trypan wash, cells are washed with PBS, fixed, and stained with DAPI (nuclear blue fluorescent label), which serves to label nuclei of cells. By a simple fluorometric quantification through plate reading of nuclear (blue) or particle (red/green) fluorescence we can examine the ratio of relative fluorescence units of green:blue and determine a phagocytic index indicative of amount of fluorescent bacteria internalized per cell. The duration of assay using a 96-well method and multichannel pipettes for washing, from end of phagocytosis to end of data acquisition, is less than 45 min. Flow cytometry could be used in a similar manner but the advantage of fluorometry is its high throughput, rapid method of assessment with minimal manipulation of samples and quick quantification of fluorescent intensity per cell. Similar strategies can be applied to non adherent cells, live labeled bacteria, actin polymerization, and essentially any process utilizing fluorescence. Therefore, fluorometry is a promising method for its low cost, high throughput capabilities in the study of cellular processes.

Here we present a protocol to quantify phagocytosis of fluorescent particles by adherent macrophage cell line using a fluorometric method. This method facilitates a high throughput quantification of particle internalization as well as the resulting actin polymerization.

高通量荧光技术巨噬细胞吞噬功能和肌动蛋白聚合的评估

The goal of fluorometric analysis is to serve as an efficient, cost effective, high throughput method of analyzing phagocytosis and other cellular ...

Measuring Granulocyte and Monocyte Phagocytosis and Oxidative Burst Activity in Human Blood

The granulocyte and monocyte phagocytosis and oxidative burst (OB) activity assay can be used to study the innate immune system. This manuscript provides the necessary methodology to add this assay to an exercise immunology arsenal. The first step in this assay is to prepare two aliquots ("H" and "F") of whole blood (heparin). Then, dihydroethidium is added to the H aliquot, and both aliquots are incubated in a warm water bath followed by a cold water bath. Next, Staphylococcus aureus (S. aureus) is added to the H aliquot and fluorescein isothiocyanate-labeled S. aureus is added to the F aliquot (bacteria:phagocyte = 8:1), and both aliquots are incubated in a warm water bath followed by a cold water bath. Then, trypan blue is added to each aliquot to quench extracellular fluorescence, and the cells are washed with phosphate-buffered saline. Next, the red blood cells are lysed, and the white blood cells are fixed. Finally, a flow cytometer and appropriate analysis software are used to measure granulocyte and monocyte phagocytosis and OB activity. This assay has been used for over 20 years. After heavy and prolonged exertion, athletes experience a significant but transient increase in phagocytosis and an extended decrease in OB activity. The post-exercise increase in phagocytosis is correlated with inflammation. In contrast to normal weight individuals, granulocyte and monocyte phagocytosis is chronically elevated in overweight and obese participants, and is modestly correlated with C-reactive protein. In summary, this flow cytometry-based assay measures the phagocytosis and OB activity of phagocytes and can be used as an additional measure of exercise- and obesity-induced inflammation.

The purpose of this manuscript is to present a method for measuring monocyte and granulocyte phagocytosis and oxidative burst activity in human blood samples.

人体血液中测粒及单核细胞吞噬功能和呼吸爆发活动

The granulocyte and monocyte phagocytosis and oxidative burst (OB) activity assay can be used to study the innate immune system. This manuscript provides ...

Time-lapse 3D Imaging of Phagocytosis by Mouse Macrophages

Phagocytosis plays a key role in host defense, as well as in tissue development and maintenance, and involves rapid, receptor-mediated rearrangements of the actin cytoskeleton to capture, envelop and engulf large particles. Although phagocytic receptors, downstream signaling pathways, and effectors, such as Rho GTPases, have been identified, the dynamic cytoskeletal remodeling of specific receptor-mediated phagocytic events remain unclear. Four decades ago, two distinct mechanisms of phagocytosis, exemplified by Fc&#947; receptor (Fc&#947;R)- and complement receptor (CR)-mediated phagocytosis, were identified using scanning electron microscopy. Binding of immunoglobulin G (IgG)-opsonized particles to Fc&#947;Rs triggers the protrusion of thin membrane extensions, which initially form a so-called phagocytic cup around the particle before it becomes completely enclosed and retracted into the cell. In contrast, complement opsonized particles appear to sink into the phagocyte following binding to complement receptors. These two modes of phagocytosis, phagocytic cup formation and sinking in, have become well established in the literature. However, the distinctions between the two modes have become blurred by reports that complement receptor-mediated phagocytosis may induce various membrane protrusions. With the availability of high resolution imaging techniques, phagocytosis assays are required that allow real-time 3D (three dimensional) visualization of how specific phagocytic receptors mediate the uptake of individual particles. More commonly used approaches for the study of phagocytosis, such as end-point assays, miss the opportunity to understand what is happening at the interface of particles and phagocytes. Here we describe phagocytic assays, using time-lapse spinning disk confocal microscopy, that allow 3D imaging of single phagocytic events. In addition, we describe assays to unambiguously image Fc&#947; receptor- or complement receptor-mediated phagocytosis.

Here we describe methods using spinning disk confocal microscopy to image single phagocytic events by mouse resident peritoneal macrophages. The protocols can be extended to other phagocytic cells.

小鼠巨噬细胞吞噬的延时3D 成像

Phagocytosis plays a key role in host defense, as well as in tissue development and maintenance, and involves rapid, receptor-mediated rearrangements of ...

Prediction of Red Blood Cell Antibody Significance Using the Monocyte-Macrophage Assay

Derived from monocytes in the bone marrow, macrophages are large, innate immune cells that play a major role in clearing dead cells, debris, tumor cells, and foreign pathogens. The phagocytic capacity of monocytes versus macrophages is a concept that is not well understood. Here, we aim to examine a difference in the phagocytosis of monocytes versus macrophages, specifically M1/M2 macrophages, against various opsonized red cells using a modified and updated version of the established monocyte monolayer assay (MMA). Peripheral blood mononuclear cells (PBMCs) were isolated from donor buffy coats. Using purified monocytes, inflammatory M1 and anti-inflammatory M2 macrophages were produced by in vitro culture and polarization. M1/M2 cells were harvested and used in an MMA-like assay, which we refer to as the M-MA, to decipher clinically significant phagocytosis of various red cell antibodies. A phagocytic index (PI) &#62; 5 was deemed clinically significant phagocytosis with the use of monocytes. A phagocytic index (PI) &#62; 12 was deemed clinically significant phagocytosis with the use of M1/M2 macrophages. M2 macrophages demonstrate an increased ability to phagocytose opsonized RBCs compared to monocytes and M1s. The same weak antibody (anti-S) yields significant phagocytosis with only M2 macrophages (PI=43) but not M1s (PI=2) or monocytes (PI=0), and this was demonstrated repeatedly using various antibodies. The use of M2 macrophages instead of monocytes may allow for more accurate results as these cells are more phagocytic, offering further clinical relevance to the assay. Further studies with different antibodies to red blood cells, including validation of the monocyte-macrophage assay (M-MA) with antibodies having known clinical significance, may show the M-MA&#160;more useful to help predict clinically significant red cell alloantibodies and transfusion reactions. This method will advance the field of transfusion medicine and immunology.

We have developed enhancements and updated methods for the existing monocyte-monolayer assay (MMA), in which macrophages are used to help better predict the clinical relevance of red cell alloantibodies in transfusion medicine and immunology. This assay is named the monocyte-macrophage assay (M-MA).

使用单核细胞-巨噬细胞测定预测红细胞抗体的意义

Derived from monocytes in the bone marrow, macrophages are large, innate immune cells that play a major role in clearing dead cells, debris, tumor cells, ...

Real-Time Imaging of Macrophage Phagocytosis of IgG-Opsonized Red Blood Cells

Source: Horsthemke, M., et al. <a href="https://app.jove.com/v/57566">Time-lapse 3D Imaging of Phagocytosis by Mouse Macrophages.</a> J. Vis. Exp. (2018)
This video demonstrates an assay to monitor independent macrophage-mediated red blood cell (RBC) phagocytosis events. Mouse immunoglobulin G (IgG)-opsonized human RBCs are incubated with mouse macrophages and observed under a confocal microscope. The RBCs are engulfed by macrophages via a phagocytic cup formation and targeted for lysosomal degradation.

IgG 调导红细胞巨噬细胞吞噬作用的实时成像

Source: Horsthemke, M., et al. Time-lapse 3D Imaging of Phagocytosis by Mouse Macrophages. J. Vis. Exp. (2018)
This video demonstrates an assay to monitor ...

JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

Specialized Assays

An In Vitro Assay for Evaluating the Immunomodulatory Impact of Monocytes on Leukocyte Proliferation

Source: Hsu, P., et al. <a href="https://app.jove.com/v/53265">Assessment of the Immunomodulatory Properties of Human Mesenchymal Stem Cells (MSCs).</a> J. Vis. Exp. (2015)
This video demonstrates an in vitro assay to explore the immunomodulatory influence of mesenchymal stem cells (MSCs) on effector T cell proliferation. The assay includes co-culturing MSCs with PBMCs to induce the monocyte population to acquire an immunomodulatory phenotype, leading to the secretion of interleukin-10 (IL-10). Following incubation with activated CD4+ T cells, the induced monocytes effectively suppress T cell proliferation.

一种用于评估单核细胞对白细胞增殖免疫调节影响的体外测定

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Density Gradient Isolation of ...

In Vitro and In Vivo Models

An Assay to Assess Phagocytosis and Oxidative Burst Activity In Granulocytes and Monocytes

Source: Meaney, M. P. et al., <a href="https://app.jove.com/v/54264">Measuring Granulocyte and Monocyte Phagocytosis and Oxidative Burst Activity in Human Blood.</a> J. Vis. Exp. (2016)
This video demonstrates the assay to confirm phagocytic activity by cells in human blood via flow cytometry. The granulocytes and monocytes confirm their phagocytic activity by showing green and red fluorescence confirming the presence of engulfed bacteria and oxidative burst activity respectively.

评估粒细胞和单核细胞吞噬作用和氧化爆发活性的测定

All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human ...

An Assay for Quantifying Antibody-Mediated Phagocytosis of Parasite-Infected Erythrocytes

Source: Quintana, M. d. P. et al., <a href="https://app.jove.com/v/61538">Measuring Naturally Acquired Phagocytosis-Inducing Antibodies to Plasmodium falciparum Parasites by a Flow Cytometry-Based Assay.</a>&#160;J. Vis. Exp.&#160;(2020)
This video demonstrates a flow cytometry-based phagocytosis assay involving labeled and opsonized Plasmodium falciparum-infected erythrocytes by monocytes. Flow cytometry identifies fluorescence in monocytes, confirming the successful identification of opsonized erythrocytes through phagocytosis.

一种定量抗体介导的寄生虫感染红细胞吞噬作用的测定

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Parasite culture
NOTE: Follow the ...

An In Vitro Assay to Measure Antibody-Dependent Cellular Phagocytosis by Phagocytes

Source: Powell, R. L., et al. <a href="https://app.jove.com/v/60149">Isolation of Leukocytes from Human Breast Milk for Use in an Antibody-dependent Cellular Phagocytosis Assay of HIV Targets.</a> J. Vis. Exp. (2019).
This video demonstrates an assay to measure the antibody-dependent cellular phagocytosis (ADCP) by breast milk phagocytes. Fluorescently labeled microspheres conjugated to an antigen-antibody complex, are combined with a cell suspension isolated from breast milk to induce antibody-dependent cellular phagocytosis by the phagocytic leukocytes. Upon labeling the cells with antibodies against common leukocyte antigen (CD45), flow cytometry is used to measure the fluorescence intensity of microsphere-positive viable phagocytic leukocytes, indicating the level of ADCP activity.

一种测量吞噬细胞抗体依赖性细胞吞噬作用的体外测定

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Target Microsphere preparation

	...