深圳和DW收到一个不受限制的资金VIROPHARMA的。

1。菠萝蛋白酶处理过的红血细胞的制备<ol><li>洗0类型的红细胞3倍用1×PBS（离心664×g离心2-5分钟）。 </li><li>孵育1卷袋装RBC与两卷的10分钟0.5％的菠萝蛋白酶溶液在37°C </li><li>洗细胞3倍用1×PBS。 </li><li>该细胞可以被存储为在4℃下在1×PBS中3％的溶液，至少一个星期。 </li></ol> 2。 C3和C4沉积分析红细胞用流式细胞仪<ol><li>通过在56℃下将样品加热30分钟热失活所需的病人血清的量（或其它抗红细胞抗体来源） </li><li>在VBG洗菠萝蛋白酶处理的红细胞三次- （5 mM的佛罗那，150 mM氯化钠，0.05％明胶pH7.4）中重悬在他们VBG + +（VBG - + 2毫摩尔MgCl 2 +的10mM的CaCl 2），得到0.5％的溶液。 </li><li>吸取以下组件成圆底板中，用玻璃珠（六或适当的混合）:25微升0.5％的红细胞，37.5微升新鲜的AB血清（补源）和37.5μL200μg/ ml的抗C5。抗C5是昂贵的并且可能是很难获得的。人们可以考虑使用C5-/C6-/C7-或C8缺乏血清代替AB血清和抗-C5。 </li><li>添加每孔患者血清的0.1-33％的终浓度。正确的差异在血清浓度每孔由于使用热灭活的AB血清。加VBG + +得到150微升/终体积良好。关闭板与酶联免疫吸附贴膜。 </li><li>孵育1.5-2小时，在37℃振荡。 </li><li>用PBS +0.5％BSA（离心664×g离心2分钟）洗涤红细胞3次。 </li><li>添加荧光标记的抗C3和抗-C 4的单克隆抗体或其Fab片段在PBS +0.5％BSA（减少凝集），以1微克/毫升的最终浓度。这里定制开发的抗-C3-的Alexa Fluor 488和抗-C4的Alexa Fluor 647的单克隆抗体被使用;成分股的选择Ë荧光标记允许同时检测C3和C4的流式细胞仪。 </li><li>孵育30-45分钟，在室温下轻轻摇动。 </li><li>洗涤红细胞3次，用PBS +0.5％BSA中。 </li><li>重悬红细胞150微升的PBS +0.5％BSA和吸取到一个新板（玻璃珠的流式细胞仪分析之前干掉）。 </li><li>进行FACS分析。独立的单细胞在分散绘图FSC-A双峰，在 ​​单一红细胞设置闸门;选择的荧光信号（ 如 FITC和APC）的相应检测通道。 </li><li>使用中值荧光强度定量结果。 </li></ol>注意:可以使用同种型对照（ 例如，荧光标记的抗IL6小鼠IgG1）。然而，建议包含一个真阴性对照，例如通过孵育红细胞无患者血清（只有AB血清）或没有补体源（热灭活的AB血清和热灭活患者血清），一ð然后用相同的抗-C3-的Alexa Fluor 488和抗-C4的Alexa Fluor 647抗体染色这些。这给出了比在红血细胞12的情况下的同种型对照更有效的控制，尽管这些不同的控件通常给予相同的，负面的结果。 3。定量溶血测定<ol><li>通过将样品加热30分钟，在56℃下热失活所需的患者的血清的量（或其它抗红细胞抗体来源） </li><li>洗涤红细胞3倍与VBG -一旦与VBG + +。 </li><li>吸取以下组件成圆底板中，用玻璃珠（对于适当的混合）:35微升3％的红细胞，将25μl新鲜AB血清（补体源）和1〜50％患者血清。正确使用热灭活的AB血清的血清浓度的差异。加VBG + +得到150微升/终体积良好。关闭板与酶联免疫吸附贴膜。 </li><li>在37°C为1.5-2小时，用颤抖。 </li><li> Çentrifuge板在664×g离心5分钟以减少减速（ 例如减速至完全停止在2-3分钟）。 </li><li>小心吸取90微升上清液在一个新的微孔板。它以防止气泡，并防止带沿完整的细胞是在此步骤中很重要。 </li><li>在合适的分光光度计测量A414/690。 </li><li>表达的裂解作为与纯净水的样品的红细胞溶胞的百分比。 </li></ol>

<ol>
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作者什么都没有透露。

上述测定是可再现的和鲁棒性。它们很容易进行，并有可能与许多样品在同一时间（在96孔板中）执行它们。因此，这种方法也将适合于一个完全自动化的系统中， 例如在ELISA机器人系统。相反，目前使用的技术，这些测定是定量的，这将有助于例如在补体抑制剂的作用的研究。此外，该解释是客观的，这是一个改进目前使用的检测。 这两种检测方法有一个关键的步骤。重要的FACS分析是门上的单个红细胞，由于凝集的红细胞会给出虚高的信号，因为它们算作一个事件。它甚至更好地防止凝集通过采用低患者血清浓度，因为低浓度仍然提供良好的信号。在溶血测定中，关键的是要小心转移上清一个折后孵化成一个新的板块，因为红细胞的结转将导致一个不可靠的信号（如意志气泡）。它建议使用0类型的红细胞在两种测定中，以防止与AB0失配补体激活的混乱。 由于它们的可靠性和健壮性这些测定法是合适的，研究补体抑制剂的疗效AIHA这表现在参考图11中，为了检测补体激活定量细微的差别。由于其灵敏度这些测定可检测补体激活的患者，其中对红细胞的补体活化强烈建议但不通过常规测定法，诸如在Coombs阴性AIHA检测到，虽然该语句仍然需要验证。它们也可以用来确定激活补体容量同种抗体（诱导例如输血，或在一个例RhD -母亲轴承的RhD +孩子）的可能有助于临床医生预报T A检测同种抗体的临床行为。

对红血细胞（红细胞）的抗体可通过红细胞表达抗原其不存在于接收者的红细胞输血诱发。这些异体抗体可能会由于须待下列输血5，补充活化严重的急性溶血性输血反应。在自身免疫性溶血性贫血（AIHA），患者有自身抗体对自身红细胞。这导致了细胞通过的IgG结合到红细胞上的脾和/或壳体的自身抗体能够通过补体受体在肝脏6,7，激活补体的吞噬细胞的Fcγ-受体的相互作用加速间隙。暴发性补体激活导致血管内溶血是罕见的，但往往是致命的。加速，补体介导的红细胞破坏引起的任何异体或自身抗体导致急性贫血，因此潜在的致命性组织缺氧。自动抗体AIHA归类在温暖和寒冷的抗体，dependin在他们结合到红细胞（37°C或更低，分别）的最佳温度克。温暖的抗体是IgG同种型通常和IgM抗体的冷抗体同型8,9。 AIHA可继发如 lymphoprolyferative疾病，结缔组织疾病，实体肿瘤，感染或药物，但在50％的案件AIHA是特发性9。 检测丙种球蛋白（ 例如 ，IgG或IgM）和补体结合到病人的红细胞是通过半定量直接抗球蛋白（Coombs）试验（DAT）装置进行的。在DAT患者红细胞温育与抗IgG或抗体C3d。红细胞凝集的现象证明了附加的补体组分或IgG结合的存在。检测异体或在患者的血清中的自身抗体是由间接抗球蛋白试验（IAT）的装置执行。 IAT中，菠萝蛋白酶处理过的试验红细胞温育与病人血清，洗涤，然后用抗-H培养乌曼IgG抗体。的情况下的红细胞已被致敏的抗红细胞抗体存在于病人血清凝集会发生。 IgM抗体对红细胞后的菠萝蛋白酶治疗试验的红细胞与患者血清孵育，将直接导致凝集。红细胞凝集的直接Coombs试验或IAT中或者由眼睛在试管中或通过在一个小Sephadex柱由大小为1分离凝集和单个红细胞将样品加载在视觉上进行评估。 另一种常用的技术来测量红细胞的补体激活是溶血测定1中，其中患者血清诱导的菠萝蛋白酶处理的红细胞10（补体-介导的），溶血的能力进行评估。测试记为阳性时，离心后的上清液染成红色由于释放血红蛋白，并认为，如果它仍然无色为负。无论是抗人球蛋白试验和溶血试验是半定量以来的最高塞鲁米稀释来表示，其中，测试仍然是阳性。 抗球蛋白试验和溶血试验是强大的分析认为在诊断常规使用。因为这些测定是半定量的，依赖于执行它们不适合研究补体活化的红细胞上细微的差别，如在评估补体抑制剂的疗效所需的测定中的技术人员的经验。因此，我们开发了两个定量检测由（自动）抗体，以确定补体激活对红细胞，我们将在本文中介绍。 首先，我们开发了一种测定法来测量补体C3和C4的激活片段对红细胞的沉积（ 图1A）。在该试验中，人的菠萝蛋白酶处理过的0型红细胞温育用热灭活的患者血清（抗红细胞抗体来源），清新AB血清（补体源）和抗C5单克隆抗体（艾库组单抗）。在这个INCubation，会发生C3和C4的沉积，如果病人血清中含有补体激活抗红细胞抗体。为了防止红细胞裂解由下游的补体激活阻断性抗-C5单克隆抗体被加入。接着，C3和C4上的红细胞沉积是通过使用荧光标记的单克隆抗体或Fab片段与C3和C4，分别进行反应的FACS检测。门上的单个红细胞是很重要的，以确保结果的可靠性。这种技术的优点包括所需要的小体积患者材料制成，在级联的早期活化补体被评估，并且该​​方法是可重复的和定量的。看着两个C3和C4的一个附加优点是可以区别的经典和凝集素途径的激活（包括C3和C4沉积）和交替途径激活（只有C3沉积）之间进行。而不是未经处理的红细胞使用菠萝蛋白酶处理的红细胞增加了作为灵敏度说。 第二个分析是基于当前使用的溶血测定（ 图1B）。人类菠萝蛋白酶治疗0型红细胞孵育用热灭活的患者血清（抗红细胞抗体源）和新鲜的AB血清（补源）。如果补体是由患者抗红细胞抗体激活，会出现剂量依赖性红细胞溶解，导致释放出血红蛋白。发布血红蛋白量是通过降低盘片的完整和支离破碎的红细胞后，测量其吸光度在上清液414 nm的量化。吸光度关联与发生溶血的量。相反，目前使用的测定法，该协议允许一个目标，溶血，这是非常可重复的和不依赖于解释该测定中的人的数量值。 这些方法的应用已在11，在那里可能使用的C1-抑制剂如AIHA补体抑制剂呈S被描述tudied。

<table><tbody><tr><td>PBS</td><td>Fresenius Kabi</td><td></td><td>&amp;nbsp;</td></tr><tr><td>BSA</td><td>Sigma</td><td>B-4287</td><td>&amp;nbsp;</td></tr><tr><td>Barbital</td><td>Fagron</td><td>0261</td><td>This chemical is subject to drug regulation.</td></tr><tr><td>Sodium Barbital</td><td>Fagron</td><td>0263</td><td>This chemical is subject to drug regulation.</td></tr><tr><td>Gelatin</td><td>Merck</td><td>1.0470.0500</td><td>&amp;nbsp;</td></tr><tr><td>Sodium chloride</td><td>Merck</td><td>1.06404.1000</td><td>&amp;nbsp;</td></tr><tr><td>Magnesium Chloride</td><td>Merck</td><td>1.05833.0250</td><td>&amp;nbsp;</td></tr><tr><td>Calcium chloride</td><td>Merck</td><td>1.0238.0500</td><td>&amp;nbsp;</td></tr><tr><td>Dylight 488 amine reactive dye</td><td>Pierce</td><td>46402</td><td>&amp;nbsp;</td></tr><tr><td>Dylight 650 amine reactive dye</td><td>Pierce</td><td>62265</td><td>&amp;nbsp;</td></tr><tr><td>&amp;alpha;C5 (Eculizumab)</td><td>Alexion Pharmaceuticals</td><td></td><td>&amp;nbsp;</td></tr><tr><td>FACS (Canto)</td><td>BD</td><td></td><td>Any FACS can be used that has the appropiate lasers.</td></tr><tr><td>Spectrophotometer (e.g. Multiskan spectrum)&amp;nbsp;</td><td>Thermo Labsystems</td><td>1500-193</td><td>Any spectrophotometer with the right wavelength range can be used</td></tr><tr><td>BD FACSDiva software v 6.1.2</td><td>BD</td><td>643629</td><td>Any compatible FACS analysis software can be used</td></tr><tr><td>Bromelain&amp;nbsp;</td><td>Sanquin</td><td>K1121</td><td>&amp;nbsp;</td></tr></tbody></table>

methods for quantitative detection of antibody-induced complement activation on red blood cells

对红血细胞（红细胞）的抗体可导致补体激活导致通过补体受体在肝脏中（血管外溶血）或导致红细胞的血管内溶解的加速间隙。同种抗体（ 如 ABO）或自身抗体红细胞抗原（如自身免疫性溶血性贫血，AIHA看出），导致补体激活有潜在危害，可-导致血管内溶解，尤其是当-致命1。目前，补体激活，由于对红细胞（自动）抗体是通过使用Coombs试验反映了补体沉积于红细胞或由非定量溶血测定反映红细胞裂解1-4评估体外 。但是，为了评估补体抑制剂的疗效，它是强制性的，具有定量技术。在这里，我们描述了两个这样的技术。首先，检测以检测C3和C4上的沉积是由抗体诱导在患者血清中红细胞是预sented。为此，FACS分析是使用荧光标记的抗-C3或抗-C 4的抗体。接着，定量溶血测定进行说明。在此测定法，补体介导的溶血引起的病人血清的测量是利用分光光度法检测所释放的血红蛋白的。这两个实验都非常重复性和定量，促进抗体诱导补体活化的研究。

图2A显示了一个代表性的散点图红细胞。对于单个红细胞合适的门可以看出，在去FSC-W - FSC-A地块（P1）。通常约95％的红细胞落入本P1栅极（单细胞）内，但是，当患者血清的高百分比被使用时，这可降低到70-80％，特别是当抗红细胞IgM是存在于病人血清。 对C4沉积AIHA患者血清的效果代表性的结果示于图2B和图2C C3沉积。如在这些图中可以看出，更多的患者血清导致更多的补体沉积，正如所预期的。奇怪的是，补体沉积发生在两个不同的正峰。这可能是不通过异质RBC群体造成的，由于红细胞​​是从单个供体绘制。我们仍在调查这一现象的原因。 图2D和2E demonstra电子温度Te的C4（ 图2D）和C3（ 图2E）的沉积测定法的重现性。他们还表现出不同的AIHA患者样本的补体沉积能力的巨大差异。 在溶血测定用含有自身抗体对红细胞的AIHA患者血清样品的代表性的结果可以看出，在图3A中 。通常情况下，与血清样品滴定得到一个不错的，可重复的曲线。病人血清有很大的不同激活补体的能力，因此执行每个病人血清的滴定。样片未经患者血清通常是由于吸收的血清样品作为补充源给出一个背景10-15％左右。因此，应小心使用足够高的患者的血清浓度，得到一个信号，即基本上是以上这样的背景。用合适的补体抑制剂如抗C5，溶血信号可以被滴定相差所示F中IGURE 3B。 <img alt="图1" fo:content-width="6in" fo:src="/files/ftp_upload/51161/51161fig1highres.jpg" src="/files/ftp_upload/51161/51161fig1.jpg" width="600px" /> 图1。的C3-和C4沉积法（A）和定量溶血测定（B）。总之，在C3-和C4沉积法的示意性概观 ，补体沉积引起的AIHA患者血清进行测定，而在溶血测定溶血诱导检测AIHA患者血清。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51161/51161fig1highres.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图2" fo:content-width="6in" fo:src="/files/ftp_upload/51161/51161fig2highres.jpg" src="/files/ftp_upload/51161/51161fig2.jpg" width="600px" /> 图2。 C3和C4的沉积有代表性的成果SSAY A）建议的门控策略。这里展示的是如何选择只为单个红细胞（P1，红色）。的凝集物示于绿色（P2）B）C4上沉积AIHA患者血清的滴定的效果。同种型对照（抗-IL-6小鼠IgG1）显示为灰色固体，同时将样品显示为黑色。增加患者的血清的量导致增加的C4沉积。C）相似为B），但现在用检测为C3沉积D）C4沉积的FACS结果显示该测定的可再现性和示出了相当大的差异的曲线图中描绘来自不同患者的血清样本之间。 Y轴表示平均荧光强度E）类似的为D），但现在的C3沉积。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51161/51161fig2highres.jpg" target="_blank">点击这里</a>查看大图。 <img alt="图3" fo:content-width="6in" fo:src="/files/ftp_upload/51161/51161fig3highres.jpg" src="/files/ftp_upload/51161/51161fig3.jpg" width="600px" /> 图3。为定量溶血测定的溶血测定A）AIHA患者血清滴定，显示效果代表了裂解重复地增加与病人血清浓度。没有裂解发生与健康供体血清（橙色;阴性对照）B）一种合适的补体抑制剂（抗C5在这种情况下）可以废除的补体介导的裂解。这里展示的是抗C5的滴定而AIHA患者血清保持在一个恒定的浓度。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51161/51161fig3highres.jpg" target="_blank">点击这里查看大图</a> 。

Watch this Scientific Journal Video about Methods for Quantitative Detection of Antibody-induced Complement Activation on Red Blood Cells at JoVE.com

Methods for Quantitative Detection of Antibody-induced Complement Activation on Red Blood Cells

在这里，我们描述了两个测定法用于测定补体活化诱导的抗红细胞。过电流测定法的主要优点是它们的数量和易于理解的性质。

定量检测抗体诱导补体激活的方法对红血细胞

Antibodies against red blood cells (RBCs) can lead to complement activation resulting in an accelerated clearance via complement receptors in the liver ...

methods-for-quantitative-detection-antibody-induced-complement

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

30.4K 次观看.  University of Amsterdam. 在这里，我们描述了两个测定法用于测定补体活化诱导的抗红细胞。过电流测定法的主要优点是它们的数量和易于理解的性质。 

视频: Methods for Quantitative Detection of Antibody-induced Complement Activation on Red Blood Cells

Measuring the 50% Haemolytic Complement (CH50) Activity of Serum

The complement system is a group of proteins that when activated lead to target cell lysis and facilitates phagocytosis through opsonisation. Individual complement components can be quantified however this does not provide any information as to the activity of the pathway. The CH50 is a screening assay for the activation of the classical complement pathway (Fig 1) and it is sensitive to the reduction, absence and/or inactivity of any component of the pathway. The CH50 tests the functional capability of serum complement components of the classical pathway to lyse sheep red blood cells (SRBC) pre-coated with rabbit anti-sheep red blood cell antibody (haemolysin). When antibody-coated SRBC are incubated with test serum, the classical pathway of complement is activated and haemolysis results. If a complement component is absent, the CH50 level will be zero; if one or more components of the classical pathway are decreased, the CH50 will be decreased. A fixed volume of optimally sensitised SRBC is added to each serum dilution. After incubation, the mixture is centrifuged and the degree of haemolysis is quantified by measuring the absorbance of the haemoglobin released into the supernatant at 540nm. The amount of complement activity is determined by examining the capacity of various dilutions of test serum to lyse antibody coated SRBC. This video outlines the experimental steps involved in analysing the level of complement activity of the classical complement pathway.

Measuring the 50% Haemolytic Complement CH50 Activity of Serum

The classical pathway is activated by antibody and culminates in target cell lysis. The CH50 assay provides a measure of the complement activity of a serum sample. This video demonstrates the steps involved in determining the CH50 of a serum sample, the calculations and interpreting of results.

测量50％的溶血性补充（CH 50）活性的血清

The complement system is a group of proteins that when activated lead to target cell lysis and facilitates phagocytosis through opsonisation. Individual ...

科研

免疫与感染

Immunostaining-Based Detection of Dynamic Alterations in Red Blood Cell Proteins

Antibody labeling of red blood cell (RBC) proteins is a commonly used, semi-quantitative method to detect changes in overall protein content or acute alterations in protein activation states. It facilitates the assessment of RBC treatments, characterization of differences in certain disease states, and description of cellular coherencies. The detection of acutely altered protein activation (e.g., through mechanotransduction) requires adequate sample preparation to preserve otherwise temporary protein modifications. The basic principle includes immobilizing the target binding sites of the desired RBC proteins to enable the initial binding of specific primary antibodies. The sample is further processed to guarantee optimal conditions for the binding of the secondary antibody to the corresponding primary antibody. The selection of non-fluorescent secondary antibodies requires additional treatment, including biotin-avidin coupling and the application of 3,3-diaminobenzidine-tetrahydrochloride (DAB) to develop the staining, which needs to be controlled in real-time under a microscope in order to stop the oxidation, and thus staining intensity, on time. For staining intensity detection, images are taken using a standard light microscope. In a modification of this protocol, a fluorescein-conjugated secondary antibody can be applied instead, which has the advantage that no further development step is necessary. This procedure, however, requires a fluorescence objective attached to a microscope for staining detection. Given the semi-quantitative nature of these methods, it is imperative to provide several control stains to account for non-specific antibody reactions and background signals. Here, we present both staining protocols and the corresponding analytical processes to compare and discuss the respective results and advantages of the different staining techniques.

Capturing dynamic changes in the protein activation of enucleated red blood cells poses methodological challenges, like the preservation of dynamic changes to acute stimuli for later assessment. The presented protocol describes sample preparation and staining techniques that enable preservation and analysis of relevant protein changes and subsequent detection.

基于免疫染色的红细胞蛋白动态改变检测

Antibody labeling of red blood cell (RBC) proteins is a commonly used, semi-quantitative method to detect changes in overall protein content or acute ...

生物化学

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.

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 ...

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 ...

Measuring Erythrocyte Complement Receptor 1 Using Flow Cytometry

CR1 (CD35, Complement Receptor type 1 for C3b/C4b) is a high molecular weight membrane glycoprotein of about 200 kDa that controls complement activation, transports immune complexes, and participates in humoral and cellular immune responses. CR1 is present on the surface of many cell types, including erythrocytes, and exhibits polymorphisms in length, structure (Knops, or KN, blood group), and density. The average density of CR1 per erythrocyte (CR1/E) is 500 molecules per erythrocyte. This density varies from one individual to another (100&#8211;1,200 CR1/E) and from one erythrocyte to another in the same individual. We present here a robust flow cytometry method to measure the density of CR1/E, including in subjects expressing a low density, with the help of an amplifying immunostaining system. This method has enabled us to show the lowering of CR1 erythrocyte expression in diseases such as Alzheimer&#39;s disease (AD), systemic lupus erythematosus (SLE), AIDS, or malaria.

The aim of this method is to determine the CR1 density in the erythrocytes of any subject by comparing with three subjects whose erythrocyte CR1 density is known. The method uses flow cytometry after immunostaining of the subjects&#39; erythrocytes by an anti-CR1 monoclonal antibody coupled to an amplified system using phycoerythrin (PE).

使用流细胞测定测量红细胞补充受体1

CR1 (CD35, Complement Receptor type 1 for C3b/C4b) is a high molecular weight membrane glycoprotein of about 200 kDa that controls complement activation, ...

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, ...

A Serum Bactericidal Assay for the Complement-Mediated Bactericidal Activity of Antibodies

Source: Weerts, H. P., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/59164">A High-throughput Shigella-specific Bactericidal Assay.</a> J. Vis. Exp. (2019).
This video demonstrates a technique to evaluate the complement-mediated bactericidal activity of antibodies present in serum. This approach combines isolated serum with pathogenic bacteria and exogenous complement proteins. Upon incubation, the bacteria are grown on an agar plate, and the produced colonies are quantified to measure the serum bactericidal titer.

补体介导的抗体杀菌活性的血清杀菌测定

Source: Weerts, H. P., et al. A High-throughput Shigella-specific Bactericidal Assay. J. Vis. Exp. (2019).
This video demonstrates a technique to evaluate ...

JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

Specialized Assays

Detection of Rhesus Factor in Human Blood Samples using Magnetic Levitation

Source: Thompson, L. et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/62550">Quantification of Cellular Densities and Antigenic Properties using Magnetic Levitation.</a>&#160;J. Vis. Exp.&#160;(2021)
This video demonstrates the detection of the Rhesus (Rh) factor in blood samples using a magnetic levitation instrument. The magnetic field aligns and levitates beads and blood cells based on density. Treating the samples with anti-Rh antibodies results in intermediate-density bead-cell complexes&#39; formation, allowing for the differentiation of Rh-positive and Rh-negative red blood cells.

使用磁悬浮法检测人血液样品中的 Rh 因子

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

Cellular Immunodiagnostics

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

Source: Powell, R. L., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/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

	...

Antibody-Based Technologies

Characterization and Functional Analysis

Measuring the Activation of Fc-Mediated Effector Functions by HA Antibodies

To assess the ability of specific antibodies of interest to target the hemagglutinin-expressing cells, replace the supernatant of the virally infected cell cultures with 25 microliters of antibody-dependent cell-mediated cytotoxicity, or ADCC, assay medium.
Next, in a separate 96-well plate, perform 4-fold dilutions of the antibodies of interest in triplicate and fresh ADCC medium starting at 10 micrograms per milliliter according to the schematic, and taking care to include the background and no antibody controls.
When all of the antibody has been diluted, transfer 25 microliters of the diluted antibody to the appropriate corresponding wells in the experimental cell plate and place the plate in the cell culture incubator. After 15 minutes, add 7.5 times 10 to the 4th modified effector Jurkat cells, expressing the appropriate Fc receptor per well in 25 microliters of ADCC medium for a final volume of 75 microliters of ADCC medium per well.
Return the plate to the cell culture incubator for 6 hours, thawing luciferase substrate buffer in a 37-degree Celsius water bath during the last hour of the incubation. When the buffer is ready, add 75 microliters of luciferase substrate buffer to all of the wells except the background control well, and read the plate on a luminometer. The fold induction of antibody-dependent cell-mediated cytotoxicity in the transfected or infected cell cultures can then be calculated.

定量检测抗体诱导补体激活的方法对红血细胞

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测量50％的溶血性补充（CH₅₀）活性的血清