笔者想感谢劳拉马修斯小姐表演的技巧和摄影郑明牧羊犬。这个项目是由南澳大学，实验室医学程序。

制备的5倍Veronal缓冲液（VBS） <ol><li>要准备Veronal缓冲液（VBS），3个单独的解决方案都需要做好准备。 </li><li>准备通过溶解在蒸馏水350毫升NaCl和0.94gm钠巴比妥21.25gm溶液1。氯化钠和巴比妥钠终浓度分别为1.02M和13MM。 </li><li>准备通过溶解在热蒸馏水125毫升巴比妥1.44gm溶液2。巴比妥最终浓度62.5mM。 </li><li>准备通过溶解在蒸馏水100ml 氯化钙氯化镁2和4.41gm 20.33gm溶液3。 2氯化镁和氯化钙2终浓度分别是218万和440MM。 </li><li>混合解决方案1和2，冷却到室温。 </li><li>一旦合并的解决方案已经冷却，加1.25毫升解决方案3，使用1M盐酸调节pH值至7.3-7.5。 </li><li>调整最终体积至500ml，用蒸馏水，准备了5倍​​原液。 </li><li>为了准备1X的工作方案，用蒸馏水稀释股票1:5。 </li></ol>致敏的绵羊红细胞溶血<ol><li>准备首先用VBS稀释1:50的溶血</li><li>至SRBC4毫升添加的VBS6毫升，轻轻颠倒混合</li><li>离心600克x5分钟</li><li>弃上清，另2次清洗细胞</li><li>最后一次洗涤后，离心900克x5分钟包细胞的细胞</li><li>弃上清，重悬细胞足够的VBS准备10％的解决方案，即细胞排列0.5毫升悬浮于5毫升的缓冲</li><li>纷飞不断滴状细胞加入同等体积的溶血素（兔抗绵羊红细胞抗体） </li><li>在30℃水浴中孵育30分钟</li><li>轻轻地混合细胞每15分钟</li><li> Sensitised SRBC可存放过夜，4 ° C </li></ol> CH 50检测<ol><li>标签系列的管子，一式两份，每份1:8，1:16，1:32，1:64和1:128。 </li><li>重复准备了一系列控制和VBS的每个测试血清两折系列稀释</li><li>在1:4（100毫升血清+300毫升VBS的），并开始样品转移到下一个标记管200毫升。 </li><li>调匀未来稀释稀释和转移200毫升之间的一个新的枪头。 </li><li>重复，直到所有的5个稀释。 </li><li>丢弃从最终的1:128稀释200毫升。 </li><li>将暂停致敏SRBC 200ml到所有的管子。 </li><li>标签两个单独的管为空白 ，并添加致敏SRBC +200毫升VBS的200毫升。这些管子将测量的VBS的SRBC自发溶解。 </li><li>标签为总裂解另外两个单独的管和致敏SRBC +200毫升蒸馏水添加200毫升。 </li><li>轻轻混合所有的管子。 </li><li>在37℃，在水浴中搅拌15分钟后30分钟。 </li><li>在1500克离心5分钟，沉积物中的红细胞样本。 </li><li>从每管转移上清百毫升以及一个96孔平底板。 </li><li>添加蒸馏水100ml，以每口井。 </li><li>阅读使用酶标仪在540nm处的吸光度的样品。 </li></ol>计算<ol><li>计算每个样本的平均吸光度</li><li>从所有样本中减去空白吸光度（自发裂解） </li><li>为每个稀释使用下列公式计算％裂解： <img src="/files/ftp_upload/1923/1923eq1.jpg" alt="方程" /></li><li>剧情与横轴上的血清稀释的比例溶解（垂直轴）。 </li><li>计算为50％的控制和测试血清（图2）溶血所需的稀释。 </li></ol>代表性的成果： 视频包括代表性的成果的一个例子。在实践中，对照血清在测试血清的同时还可以运行，并以同样的方式对待。如下（表1）是从测试的血清样本的样本数据。数据是根据5.3方程操纵。 <table border="1"><tbody><tr><td> 样品 </td><td> 外径540 </td><td> 平均 </td><td></td><td></td></tr><tr><td>空</td><td> 0.042，0.044 </td><td> 0.043 </td><td></td><td></td></tr><tr><td>共有裂解</td><td> 0.183，0.183 </td><td> 0.183 </td><td></td><td></td></tr><tr><td colspan="5"></td></tr><tr><td> 稀释 </td><td> 外径540 </td><td> 平均 </td><td> 平均空白 </td><td> ％裂解 </td></tr><tr><td> 1:8 </td><td> 0.200，0.219 </td><td> 0.210 </td><td> 0.168 </td><td> 116 </td></tr><tr><td> 1:16 </td><td> 0.179，0.173 </td><td> 0.176 </td><td> 0.134 </td><td> 93 </td></tr><tr><td> 1:32 </td><td> 0.134，0.110 </td><td> 0.122 </td><td> 0.08 </td><td> 55 </td></tr><tr><td> 1:64 </td><td> 0.053，0.066 </td><td> 0.059 </td><td> 0.017 </td><td> 12 </td></tr><tr><td> 1:128 </td><td> 0.044，0.045 </td><td> 0.045 </td><td> 0.003 </td><td> 2 </td></tr></tbody></table><img src="http://www.jove.com/files/ftp_upload/1923/1923fig1.jpg" alt="图1" /> 图1。激活经典补体途径激活经典途径是通过结合免疫球蛋白M（IgM）的免疫球蛋白- G（IgG）的目标细胞的表面上。的抗体的Fc片段结合补体C1q，C1R激活，而这又反过来激活另一个C1R分子一起激活两个C1s的分子。 C1s的切割公开为C2的结合位点，这也是裂解C4。 C4B和C2A结合，形成一个复杂的简称为C3转化。现在这种复杂的裂解C3形成C3a和C3b，其中一些与C3形成了C5的convertase convertase结合。现在这种复杂的行为对C5的，绑定到C6开始形成膜攻击复合物（MAC），由此产生C5b。 C5b6行为，C7，打开C8的行为，并最终C9形成最终的MAC。 （Goldsby 等 ，2003）。 <img src="http://www.jove.com/files/ftp_upload/1923/1923fig2.jpg" alt="图2" /> 图2。绘制样本数据，计算控制和检测血清样本的CH50（A），绘制对稀释倍数计算的百分比（％）控制（•）和试验（+）血清样品的裂解。 （二）要计算50％的裂解，一条线是来自50％的价值，直到它与图线相交，然后绘制一条垂直线下来的稀释。在这个例子中，需要控制和21.6倍稀释的待测血清稀释35倍，达到50％溶解。这一数据表明，有少补测试血清中比对照，因为它需要较少的稀释前达到50％裂解。控制样本也显示&gt; 100％，在1:8稀释溶解。这是最有可能是由于不完全溶解的SRBC蒸馏水和补体成分更高效裂解。

<ol>
	<li>Goldsby, R. A., Kindt, T. J., Osborne, B. A., Kuby, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Immunology. , (2003).</li></ol>

CH 50含量是受到很多干扰。有些SRBC的是比其他人更脆弱，导致自发溶血补体活性无关。兔抗体的亲和力很多很多，从生产厂家之一到另一个不同，这影响的SRBC结合的抗体量。此外，敏与在细胞与不同数量的抗体涂层的SRBC的抗体结果SRBC的过程。标本采集和存储是一个重要的潜在的错误来源。补体成分如补体C1q，C3，C4和C5是非常不稳定的，因此适当的样品处理是至关重要的。长时间暴露在?...

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		<th>Material Name</th>
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		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>Sheep red blood cells</td>
<td>&nbsp;</td>
<td>CSL</td>
<td>2490201</td>
<td>Use at 1% final</td>
</tr>
<tr>
<td>Serum</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Human serum</td>
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<td>Rabbit Anti-Sheep Haemolytic serum (RBCs), Unconjugated</td>
<td>&nbsp;</td>
<td>AdB Serotec</td>
<td>C12HSB</td>
<td>&nbsp;</td>
<tr>
<td>96 well flat bottom plate</td>
<td>&nbsp;</td>
<td>Sarstedt</td>
<td>83.1839</td>
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<td>Veronal buffered saline</td>
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<td>37&deg;C room/incubator</td>
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measuring the 50% haemolytic complement (ch50) activity of serum

补体系统是一组蛋白质，当激活，导致靶细胞裂解，促进吞噬通过opsonisation。个人补体成分是可以量化的，但这不提供任何信息的途径的活动。这架CH<sub&gt; 50</sub&gt;是激活经典补体途径（图1）筛选实验，它是敏感的任何组件的通路减少，缺乏和/或闲置。这架CH<sub&gt; 50</sub&gt;测试的经典途径裂解绵羊红细胞（SRBC），兔抗绵羊红细胞抗体（溶血）预涂血清补体成分的功能性。当抗体包被的SRBC培养与血清测试，激活补体经典途径和溶血结果。如果缺少的一个补充部分，在CH<sub&gt; 50</sub&gt;等级将为零;如果一个或多个组件的经典途径是减少，在CH<sub&gt; 50</sub&gt;会有所下降。一个固定体积的最佳致敏SRBC被添加到每个血清稀释。孵育后，将混合物离心和量化的溶血程度是通过测量血红蛋白释放到上清液于540nm处的吸光度。补体活性的金额是由研究能力的各种测试血清稀释溶解抗体包被的SRBC。此视频概述参与分析经典补体途径的补充活动水平的实验步骤。

Watch this Scientific Journal Video about Measuring the 50% Haemolytic Complement CH50 Activity of Serum at JoVE.com

Measuring the 50% Haemolytic Complement CH50 Activity of Serum

经典途径激活抗体和靶细胞裂解高潮。这架CH<sub&gt; 50</sub&gt;检测提供了一个补体活性的血清样本的措施。本视频演示的CH在确定所涉及的步骤<sub&gt; 50</sub&gt;血清样本，计算和解释结果。

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 ...

measuring-the-50-haemolytic-complement-ch50-activity-of-serum

Research

JoVE Journal

Immunology and Infection

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

37.5K 次观看.  University of South Australia. 经典途径激活抗体和靶细胞裂解高潮。这架CH 50检测提供了一个补体活性的血清样本的措施。本视频演示的CH在确定所涉及的步骤 50血清样本，计算和解释结果。

视频: Measuring the 50% Haemolytic Complement CH50 Activity of Serum

Depletion of Specific Cell Populations by Complement Depletion

The purification of immune cell populations is often required in order to study their unique functions. In particular, molecular approaches such as real-time PCR and microarray analysis require the isolation of cell populations with high purity. Commonly used purification strategies include fluorescent activated cell sorting (FACS), magnetic bead separation and complement depletion. Of the three strategies, complement depletion offers the advantages of being fast, inexpensive, gentle on the cells and a high cell yield. The complement system is composed of a large number of plasma proteins that when activated initiate a proteolytic cascade culminating in the formation of a membrane-attack complex that forms a pore on a cell surface resulting in cell death1. The classical pathway is activated by IgM and IgG antibodies and was first described as a mechanism for killing bacteria. With the generation of monoclonal antibodies (mAb), the complement cascade can be used to lyse any cell population in an antigen-specific manner. Depletion of cells by the complement cascade is achieved by the addition of complement fixing antigen-specific antibodies and rabbit complement to the starting cell population. The cells are incubated for one hour at 37&deg;C and the lysed cells are subsequently removed by two rounds of washing. MAb with a high efficiency for complement fixation typically deplete 95-100% of the targeted cell population. Depending on the purification strategy for the targeted cell population, complement depletion can be used for cell purification or for the enrichment of cell populations that then can be further purified by a subsequent method.

To effectively study the function of immune cell populations their purification is often required. Complement depletion is a fast and inexpensive technique for the isolation of immune cell populations with high purity.

补消耗的特定细胞群的枯竭

The purification of immune cell populations is often required in order to study their unique functions.  In particular, molecular approaches such as ...

科研

免疫与感染

Determining the Reactivity and Titre of Serum using a Haemagglutination Assay

Haemagglutination is a specific form of agglutination and is used when antibodies bind to red blood cells, which act as a particulate antigen. Red blood cells are particularly useful targets as they are readily available and agglutination is observable using the naked eye. This technique is commonly used to determine the titre of an antibody (Ab), for blood grouping and viral quantification. In this video, the steps involved in preparing and performing a haemagglutination assay is demonstrated using antibodies specific to blood group A-antigens added to red blood cells (Revercells). The antiserum is serially diluted in a 96 well U-bottom microtitre tray, to which is added a suspension of Revercells. The samples are mixed and then incubated at 37&deg;C for 60 minutes. After this time, the samples can then be easily scored for  ve, +ve and intermediate (-/+) haemagglutination reactions. This approach allows for the reactivity and titre of a serum sample to be assessed using a rapid and simple technique. The video will cover the theory behind the assay, how the results are read and interpreted, how the titre is determined, how the assay can be modified and any issues associated with the use of this technique.

Haemagglutination is a form of agglutination where antibodies bind to red blood cells. Red blood cells are both readily available and the results are readily observable using the naked eye. This video demonstrates the steps involved in a haemagglutination assay, interpreting the results and determining the titre.

确定使用血凝检测血清中的反应和滴度

Haemagglutination is a specific form of agglutination and is used when antibodies bind to red blood cells, which act as a particulate antigen. Red blood ...

Measuring Calpain Activity in Fixed and Living Cells by Flow Cytometry

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, including signaling, cytoskeletal remodeling, regulation of gene expression, apoptosis and cell cycle progression 1. Calpains have been implicated in many pathologies including muscular dystrophies, cancer, diabetes, Alzheimer's disease and multiple sclerosis 1. Calpain regulation is complex and incompletely understood. mRNA and protein levels correlate poorly with activity, limiting the use of gene or protein expression techniques to measure calpain activity. This video protocol details a flow cytometric assay developed in our laboratory for measuring calpain activity in fixed and living cells. This method uses the fluorescent substrate BOC-LM-CMAC, which is cleaved specifically by calpain, to measure calpain activity. 2 In this video, calpain activity in fixed and living murine 32Dkit leukemia cells, alone or as part of a splenocyte population is measured using an LSRII (BD Bioscience). 32Dkit cells are shown to have elevated activity compared to normal splenocytes.

This article will detail the protocol for measuring calpain activity in fixed and living cells using flow cytometry.

在固定和活细胞，流式细胞仪测量钙蛋白酶活性

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, ...

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of Listeria, which results in systemic infection2. After injection, Listeria quickly disseminates to the spleen and liver due to uptake by CD8&alpha;+ dendritic cells and Kupffer cells3,4. Once phagocytosed, various bacterial proteins enable Listeria to escape the phagosome, survive within the cytosol, and infect neighboring cells5. During the first three days of infection, different innate immune cells (e.g. monocytes, neutrophils, NK cells, dendritic cells) mediate bactericidal mechanisms that minimize Listeria proliferation. CD8+ T cells are subsequently recruited and responsible for the eventual clearance of Listeria from the host, typically within 10 days of infection6.
Successful clearance of Listeria from infected mice depends on the appropriate onset of host immune responses6	. There is a broad range of sensitivities amongst inbred mouse strains7,8. Generally, mice with increased susceptibility to Listeria infection are less able to control bacterial proliferation, demonstrating increased bacterial load and/or delayed clearance compared to resistant mice. Genetic studies, including linkage analyses and knockout mouse strains, have identified various genes for which sequence variation affects host responses to Listeria infection6,8-14. Determination and comparison of infection kinetics between different mouse strains is therefore an important method for identifying host genetic factors that contribute to immune responses against Listeria. Comparison of host responses to different Listeria strains is also an effective way to identify bacterial virulence factors that may serve as potential targets for antibiotic therapy or vaccine design.
We describe here a straightforward method for measuring bacterial load (colony forming units [CFU] per tissue) and preparing single-cell suspensions of the liver and spleen for FACS analysis of immune responses in Listeria-infected mice. This method is particularly useful for initial characterization of Listeria infection in novel mouse strains, as well as comparison of immune responses between different mouse strains infected with Listeria. We use the Listeria monocytogenes EGD strain15 that, when cultured on blood agar, exhibits a characteristic halo zone around each colony due to &beta;-hemolysis1 (Figure 1). Bacterial load and immune responses can be determined at any time-point after infection by culturing tissue homogenate on blood agar plates and preparing tissue cell suspensions for FACS analysis using the protocols described below. We would note that individuals who are immunocompromised or pregnant should not handle Listeria, and the relevant institutional biosafety committee and animal facility management should be consulted before work commences.

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes is a model organism for studying immune responses and genetic susceptibility to intracellular bacteria in mice. This method enables one to measure bacterial load and generate single-cell suspensions of the liver and spleen from mice for FACS analysis to determine changes in immune cells due to Listeria infection.

测量细菌感染小鼠和免疫反应李斯特菌</em

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of ...

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 ...

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne relapsing fever (Borrelia spp.), Rocky Mountain Spotted fever (Rickettsia rickettsii), ehrlichiosis (Ehrlichia chaffeensis and E. equi), anaplasmosis (Anaplasma phagocytophilum), encephalitis (tick-borne encephalitis virus), babesiosis (Babesia spp.), Colorado tick fever (Coltivirus), and tularemia (Francisella tularensis) 1-8. To be properly transmitted into the host these infectious agents differentially regulate gene expression, interact with tick proteins, and migrate through the tick 3,9-13. For example, the Lyme disease agent, Borrelia burgdorferi, adapts through differential gene expression to the feast and famine stages of the tick's enzootic cycle 14,15. Furthermore, as an Ixodes tick consumes a bloodmeal Borrelia replicate and migrate from the midgut into the hemocoel, where they travel to the salivary glands and are transmitted into the host with the expelled saliva 9,16-19.
As a tick feeds the host typically responds with a strong hemostatic and innate immune response 11,13,20-22. Despite these host responses, I. scapularis can feed for several days because tick saliva contains proteins that are immunomodulatory, lytic agents, anticoagulants, and fibrinolysins to aid the tick feeding 3,11,20,21,23. The immunomodulatory activities possessed by tick saliva or salivary gland extract (SGE) facilitate transmission, proliferation, and dissemination of numerous tick-borne pathogens 3,20,24-27. To further understand how tick-borne infectious agents cause disease it is essential to dissect actively feeding ticks and collect tick saliva. This video protocol demonstrates dissection techniques for the collection of hemolymph and the removal of salivary glands from actively feeding I. scapularis nymphs after 48 and 72 hours post mouse placement. We also demonstrate saliva collection from an adult female I. scapularis tick.

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

The collection of infected tick hemolymph, salivary glands, and saliva is important to study how tick-borne pathogens cause disease. In this protocol we demonstrate how to collect hemolymph and salivary glands from feeding Ixodes scapularis nymphs. We also demonstrate saliva collection from female I. scapularis adults.

唾液，唾液腺，淋巴收集肩突硬蜱蜱

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne ...

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

Antibodies against red blood cells (RBCs) can lead to complement activation resulting in an accelerated clearance via complement receptors in the liver (extravascular hemolysis) or leading to intravascular lysis of RBCs. Alloantibodies (e.g. ABO) or autoantibodies to RBC antigens (as seen in autoimmune hemolytic anemia, AIHA) leading to complement activation are potentially harmful and can be - especially when leading to intravascular lysis - fatal1. Currently, complement activation due to (auto)-antibodies on RBCs is assessed in vitro by using the Coombs test reflecting complement deposition on RBC or by a nonquantitative hemolytic assay reflecting RBC lysis1-4. However, to assess the efficacy of complement inhibitors, it is mandatory to have quantitative techniques. Here we describe two such techniques. First, an assay to detect C3 and C4 deposition on red blood cells that is induced by antibodies in patient serum is presented. For this, FACS analysis is used with fluorescently labeled anti-C3 or anti-C4 antibodies. Next, a quantitative hemolytic assay is described. In this assay, complement-mediated hemolysis induced by patient serum is measured making use of spectrophotometric&#160;detection of the released hemoglobin. Both of these assays are very reproducible and quantitative, facilitating studies of antibody-induced complement activation.

Here we describe two assays for measuring complement activation induced by antibodies against red blood cells. The major advantage over the current assays is their quantitative and easy-to-interpret nature.

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 ...

Measuring Local Anaphylaxis in Mice

Allergic responses are the result of the activation of mast cells and basophils, and the subsequent release of vasoactive and proinflammatory mediators. Exposure to an allergen in a sensitized individual can result in clinical symptoms that vary from minor erythema to life threatening anaphylaxis. In the laboratory, various animal models have been developed to understand the mechanisms driving allergic responses. Herein, we describe a detailed method for measuring changes in vascular permeability to quantify localized allergic responses. The local anaphylaxis assay was first reported in the 1920s, and has been adapted from the technique published by Kojima et al. in 20071. In this assay, mice sensitized to OVA are challenged in the left ear with vehicle and in the right ear with OVA. This is followed by an intravenous injection of Evans Blue dye. Ten min after injecting Evans Blue, the animal is euthanized and the dye that has extravasated into the ears is extracted overnight in formamide. The absorbance of the extracted dye is then quantified with a spectrophotometer. This method reliably results in a visual and quantifiable manifestation of a local allergic response.

Allergic responses, characterized by the activation of mast cells and basophils, are driven by the cross-linking of IgE and release of proinflammatory mediators. A quantitative assessment of allergic responses can be achieved by using Evans Blue dye to monitor changes in vascular permeability after allergen challenge.

小鼠量度本地过敏反应

Allergic responses are the result of the activation of mast cells and basophils, and the subsequent release of vasoactive and proinflammatory mediators. ...

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 ...

Assays for Studying the Role of Vitronectin in Bacterial Adhesion and Serum Resistance

Bacteria utilize complement regulators as a means of evading the host immune response. Here, we describe protocols for evaluating the role vitronectin acquisition at the bacterial cell surface plays in resistance to the host immune system. Flow cytometry experiments identified human plasma vitronectin as a ligand for the bacterial receptor outer membrane protein H of Haemophilus influenzae type f. An enzyme-linked immunosorbent assay was employed to characterize the protein-protein interactions between purified recombinant protein H and vitronectin, and binding affinity was assessed using bio-layer interferometry. The biological importance of the binding of vitronectin to protein H at the bacterial cell surface in evasion of the host immune response was confirmed using a serum resistance assay with normal and vitronectin-depleted human serum. The importance of vitronectin in bacterial adherence was analyzed using glass slides with and without vitronectin coating, followed by Gram staining. Finally, bacterial adhesion to human alveolar epithelial cell monolayers was investigated. The protocols described here can be easily adapted to the study of any bacterial species of interest.

This report describes protocols for characterizing interactions between bacterial outer membrane proteins and the human complement regulator vitronectin. The protocols can be used to study the binding reactions and biological function of vitronectin in any bacterial species.

Vitronectin 在细菌黏附和血清耐药性中作用的实验研究

Bacteria utilize complement regulators as a means of evading the host immune response. Here, we describe protocols for evaluating the role vitronectin ...