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。氯化钠和钠巴比妥终浓度分别为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毫升溶液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>稀释的Revercells <ol><li>轻轻重悬的颠倒Revercells的股票解决方案，然后取出在600克1毫升的细胞和离心5分钟，在4 ° C。 </li><li>离心后，小心取出上清液，重悬细胞在1X3毫升Veronal缓冲液。 </li><li>现在可以将这些细胞保存在4 ° C，直到使用。 </li></ol>程序<ol><li> 96以及ü底板行A1 - A12转移到1X的VBS50毫升。 </li><li>孔A1，添加抗的血清50μL调匀使用吸管。 </li><li>使用一个新的枪头，删除以及A1液50μL和转让以及A2。调匀，重复这一过程，直到您到达以及A11和然后放弃这口井从去年50μL。 </li><li>血清样品现已连续稀释2倍，从1:2到1:2048。无血清添加到井A12，因为这是VBS的阴性对照。 </li><li>如果有多个血清样本进行评估，重复步骤B行等4.1-4.4 </li><li>一旦所有的稀释已添加1％Revercells50μL所有油井。注：Revercells将定居随着时间的推移，应轻轻暂停配发入井前。 </li><li>混合样品的微量托盘一侧轻轻拍打，盖，血凝反应进行1H在37 ° C可用于孵化器或适当加热室。 </li><li>孵化，从孵化器小心地取出托盘和检查血凝板。 VBS的控制以及应很好研究的。如果反应是否定的，那么结果是有效的。如果VBS示例显示了一个积极血凝结果，然后将结果不能被使用过程中应反复。积极血凝反应会出现广泛负债表上的良好的基础，而消极的反应将作为一个小型的浓缩颗粒细胞出现在井的中心。中间结果将阳性细胞与颗粒细胞的核心的光环。血清有一定的抗体，可以反应这种结果发生，但AB的不足造成一个完整的反应。 </li><li>部分代表结果预期的结果有两个图表交涉。结果无效，并有效设置的一个例子。的抗血清的滴度也将确定。 </li></ol>代表性的成果： 视频包括代表性的成果的一个例子。下面（图1，图2）是在图表的方式显示的两个例子。第一个例子是一个不能被解释为VBS的控制是积极的反应，而第二个是一个有效的检测，要确定的滴度。 <img src="/files/ftp_upload/1752/1752fig1.jpg" alt="图1" /> 图1血凝的一个例子 。阳性结果失败。在上面的图解，heamagglutination反应是不能接受的。 VBS的血清阴性对照，显示了积极的血凝反应，由表形成良好的基础。这表明发生了错误和不能接受的结果。在这种情况下，这个过程应该是重复的。 <img src="/files/ftp_upload/1752/1752fig2.jpg" alt="图2" /> 图2：一个积极的结果已经正确的血凝的一个例子。在图形表示，heamagglutination反应是有效的。的VBS阴性对照显示负血凝反应小号证明的沉淀，已形成良好的基础。最后积极井是第7行，这表明，此血清样本的滴度为128（即1 / 128的倒数）。这将是一种抗体，是存在于血清中适量的例子。

血凝检测是一个非常简单的的技术，允许大量的血清样本，将在很短的时间内评估。它还具有无需要专门的设备读取的优势，与合适的眼睛。这种技术也被修改为检测尿中的人绒毛膜促性腺激素激素，作为一个血凝抑制试验的一部分。在有关标准的血凝试验，有一个需要注意的是为了正确执行检测的几个领域。首先，这项技术是基于一个凹面上的细胞沉淀。因此，至关重要的是，使用一个U型或圆...

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<td>Revercells</td>
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<td>Veronal buffered saline</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Use at 1x final</td>
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determining the reactivity and titre of serum using a haemagglutination assay

血凝是一种特定的凝集的形式，使用时红血细胞，它作为颗粒性抗原抗体结合。红血细胞是特别有用的目标，因为他们都是现成的，是用肉眼观察凝集。这种技术常用来确定的抗体滴度（AB），血型和病毒定量。在这个视频，在编制和执行血凝检测所涉及的步骤演示使用一个抗原添加到红血细胞（Revercells）血型特异性抗体。抗血清稀释96以及U型底酶标托盘，这是增加了一个悬挂的Revercells的。样本混合在一起，然后在37 ° C孵育60分钟。过了这段时间，样品可以很容易地取得VE，+ ve和中间的(-/+)血凝反应。此方法允许使用一个快速和简单的的技术进行评估血清样品的活性和滴度。视频将覆盖检测背后的理论，如何理解和解释的结果是，如何确定滴度是可以修改，如何检测和使用这项技术与相关的任何问题。

Watch this Scientific Journal Video about Determining the Reactivity and Titre of Serum using a Haemagglutination Assay at JoVE.com

Determining the Reactivity and Titre of Serum using a Haemagglutination Assay

血凝是抗体结合到红细胞的凝集形成。红血细胞都是现成的，结果很容易用肉眼观察。本视频演示了在血凝实验所涉及的步骤，解释的结果，并确定滴度。

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 ...

determining-reactivity-titre-serum-using-haemagglutination

Research

JoVE Journal

Immunology and Infection

20.9K 次观看.  University of South Australia. 血凝是抗体结合到红细胞的凝集形成。红血细胞都是现成的，结果很容易用肉眼观察。本视频演示了在血凝实验所涉及的步骤，解释的结果，并确定滴度。

视频: Determining the Reactivity and Titre of Serum using a Haemagglutination Assay

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

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 ...

Determining Optimal Cytotoxic Activity of Human Her2neu Specific CD8 T cells by Comparing the Cr51 Release Assay to the xCELLigence System

Cytotoxic CD8 T cells constitute a subgroup of T cells that are capable of inducing the death of infected or malignant host cells1. These cells express a specialized receptor, called the T cell receptor (TCR), which can recognize a specific antigenic peptide bound to HLA class I molecules2. Engagement of infected cells or tumor cells through their HLA class I molecule results in production of lytic molecules such as granzymes and perforin resulting in target cell death. While it is useful to determine frequencies of antigen-specific CD8 T cells using assays such as the ELIspot or flow cytometry, it is also helpful to ascertain the strength of CD8 T cell responses using cytotoxicity assays3. The most recognizable assay for assessing cytotoxic function is the Chromium Release Assay (CRA), which is considered a standard assay 4. The CRA has several limitations, including exposure of cells to gamma radiation, lack of reproducibility, and a requirement for large numbers of cells. Over the past decade, there has been interest in adopting new strategies to overcome these limitations. Newer approaches include those that measure caspase 
release 4, BLT esterase activity 5 and surface expression of CD107 6. The impedance-based assay, using the Roche xCelligence system, was examined in the present paper for its potential as an alternative to the CRA. Impedance or opposition to an electric current occurs when adherent tumor cells bind to electrode plates. Tumor cells detach following killing and electrical impedance is reduced which can be measured by the xCelligence system. The ability to adapt the impedance-based approach to assess cell-mediated killing rests on the observation that T cells do not adhere tightly to most surfaces and do not appear to have much impact on impedance thus diminishing any concern of direct interference of the T cells with the measurement. Results show that the impedance-based assay can detect changes in the levels of antigen-specific cytotoxic CD8 T cells with increased sensitivity relative to the standard CRA. Based on these results, impedance-based approaches may be good alternatives to CRAs or other approaches that aim to measure cytotoxic CD8 T cell functionality.

The chromium release assay, a common assay for detecting cytotoxic T cell activity, has several limitations. Using antigen-specific CD8 T cells and the human breast cancer tumor line, SKBR3, in the present article, an impedance-based approach was examined for the capability of detecting cell killing.

确定最佳人力Her2neu特异性CD8 + T细胞的细胞毒活性的比较CR51发行含量xCELLigence系统

Cytotoxic CD8 T cells constitute a subgroup of T  cells that are capable of inducing the death of infected or malignant host  cells1. These cells express ...

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological deficiencies. GVHD is caused by mature alloreactive T cells present in the bone marrow graft that are infused into the recipient and cause damage to host organs. However, in mice, T cells must be added to the bone marrow inoculum to cause GVHD. Although extensive work has been done to characterize T cell responses post transplant, bioluminescent imaging technology is a non-invasive method to monitor T cell trafficking patterns in vivo. 
Following lethal irradiation, recipient mice are transplanted with bone marrow cells and splenocytes from donor mice. T cell subsets from L2G85.B6 (transgenic mice that constitutively express luciferase) are included in the transplant. By only transplanting certain T cell subsets, one is able to track specific T cell subsets in vivo, and based on their location, develop hypotheses regarding the role of specific T cell subsets in promoting GVHD at various time points. At predetermined intervals post transplant, recipient mice are imaged using a Xenogen IVIS CCD camera. Light intensity can be quantified using Living Image software to generate a pseudo-color image based on photon intensity (red = high intensity, violet = low intensity). 
Between 4-7 days post transplant, recipient mice begin to show clinical signs of GVHD. Cooke et al.1 developed a scoring system to quantitate disease progression based on the recipient mice fur texture, skin integrity, activity, weight loss, and posture. Mice are scored daily, and euthanized when they become moribund. Recipient mice generally become moribund 20-30 days post transplant. 
Murine models are valuable tools for studying the immunology of GVHD. Selectively transplanting particular T cell subsets allows for careful identification of the roles each subset plays. Non-invasively tracking T cell responses in vivo adds another layer of value to murine GVHD models.

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Murine bone marrow transplantation is a widely used technique to study immunological mechanisms governing graft-versus-host disease in humans. The ability to monitor T cell trafficking patterns in vivo allows for detailed analysis of the development and perpetuation of T cell responses during graft-versus-host disease.

移植物抗宿主病的诱导和<em在体内</em&gt; T细胞监测中的应用MHC匹配的小鼠模型

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological ...

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a prerequisite for pneumococcal infections such as pneumonia and otitis media. In vitro adherence assays can be used to study the attachment of pneumococci to epithelial cell monolayers and to investigate potential interventions, such as the use of probiotics, to inhibit pneumococcal&#160;colonization.&#160;The protocol described here is used to investigate the effects of the probiotic Streptococcus salivarius on the adherence of pneumococci to the human epithelial cell line CCL-23 (sometimes referred to as HEp-2 cells). The assay involves three main steps: 1) preparation of epithelial and bacterial cells, 2) addition of bacteria to epithelial cell monolayers, and 3) detection of adherent pneumococci by viable counts (serial dilution and plating) or quantitative real-time PCR (qPCR). This technique is relatively straightforward and does not require specialized equipment other than a tissue culture setup. The assay can be used to test other probiotic species and/or potential inhibitors of pneumococcal colonization and can be easily modified to address other scientific questions regarding pneumococcal adherence and invasion.

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

In vitro adherence assays can be used to study the attachment of Streptococcus pneumoniae to epithelial cell monolayers and to investigate potential interventions such as the use of probiotics for inhibiting pneumococcal colonization.

使用一个调查益生素对肺炎球菌定植的影响<em&gt;体外</em&gt;粘附试验

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a ...

Pairwise Growth Competition Assay for Determining the Replication Fitness of Human Immunodeficiency Viruses

In vitro fitness assays are essential tools for determining viral replication fitness for viruses such as HIV-1. Various measurements have been used to extrapolate viral replication fitness, ranging from the number of viral particles per infectious unit, growth rate in cell culture, and relative fitness derived from multiple-cycle growth competition assays. Growth competition assays provide a particularly sensitive measurement of fitness since the viruses are competing for cellular targets under identical growth conditions. There are several experimental factors to consider when conducting growth competition assays, including the multiplicity of infection (MOI), sampling times, and viral detection and fitness calculation methods. Each factor can affect the end result and hence must be considered carefully during the experimental design. The protocol presented here includes steps from constructing a new recombinant HIV-1 clone to performing growth competition assays and analyzing the experimental results. This protocol utilizes experimental parameter values previously shown to yield consistent and robust results. Alternatives are discussed, as some parameters need to be adjusted according to the cell type and viruses being studied. The protocol contains two alternative viral detection methods to provide flexibility as the availability of instruments, reagents and expertise varies between laboratories.

Growth competition between nearly isogenic viruses provides a sensitive measurement for determining relative replication fitness. The protocols described here include the construction of recombinant HIV-1 clones, virus propagation and growth competition and analysis methods optimized to yield sensitive and consistent results.

成对增长竞争测定法测定人类免疫缺陷病毒的复制健身

In vitro fitness assays are essential tools for determining viral replication fitness for viruses such as HIV-1. Various measurements have been used to ...

High-throughput Quantitative Real-time RT-PCR Assay for Determining Expression Profiles of Types I and III Interferon Subtypes

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific locked nucleic acid (LNA) and molecular beacon (MB) probes, transcript standards, automated multichannel pipetting, and plate drying. Determining expression among the type I interferons (IFN), especially the twelve IFN-&#945; subtypes, is limited by their shared sequence identity; likewise, the sequences of the type III IFN, especially IFN-&#955;2 and -&#955;3, are highly similar. This assay provides a reliable, reproducible, and relatively inexpensive means to analyze the expression of the seventeen interferon subtype transcripts.

This protocol describes a high-throughput qRT-PCR assay for the analysis of type I and III IFN expression signatures. The assay discriminates single base pair differences between the highly similar transcripts of these genes. Through batch assembly and robotic pipetting, the assays are consistent and reproducible.

高通量实时定量RT-PCR检测方法的确定I型和III型干扰素亚型的表达谱

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific ...

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, including phagocytosis of damaged synapses or cells, debris, and/or invading pathogens. Impaired phagocytic function has been implicated in the pathogenesis of diseases such as Alzheimer's and age-related macular degeneration, where amyloid-&#946; plaque and drusen accumulate, respectively. Despite its importance, microglial phagocytosis has been challenging to assess in vivo. Here, we describe a simple, yet robust, technique for precisely monitoring and quantifying the in vivo phagocytic potential of retinal microglia. Previous methods have relied on immunohistochemical staining and imaging techniques. Our method uses flow cytometry to measure microglial uptake of fluorescently labeled particles after intravitreal delivery to the eye in live rodents. This method replaces conventional practices that involve laborious tissue sectioning, immunostaining, and imaging, allowing for more precise quantification of microglia phagocytic function in just under six hours. This procedure can also be adapted to test how various compounds alter microglial phagocytosis in physiological settings. While this technique was developed in the eye, its use is not limited to vision research.

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglial phagocytosis is critical for the maintenance of tissue homeostasis and inadequate phagocytic function has been implicated in pathology. However, assessing microglia function in vivo is technically challenging. We have developed a simple but robust technique for precisely monitoring and quantifying the phagocytic potential of microglia in a physiological setting.

评估视网膜小胶质细胞吞噬功能<em&gt;在体内</em&gt;使用基于流式细胞术流量测定

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, ...

Optimization of a Quantitative Micro-neutralization Assay

The micro-neutralization (MN) assay is a standard technique for measuring the infectivity of the influenza virus and the inhibition of virus replication. In this study, we present the protocol of an imaging-based MN assay to quantify the true antigenic relationships between viruses. Unlike typical plaque reduction assays that rely on visible plaques, this assay quantitates the entire infected cell population of each well. The protocol matches the virus type or subtype with the selection of cell lines to achieve maximum infectivity, which enhances sample contrast during imaging and image processing. The introduction of quantitative titration defines the amount of input viruses of neutralization and enables the results from different experiments to be comparable. The imaging setup with a flatbed scanner and free downloadable software makes the approach high throughput, cost effective, user friendly, and easy to deploy in most laboratories. Our study demonstrates that the improved MN assay works well with the current circulating influenza A(H1N1)pdm09, A(H3N2), and B viruses, without being significantly influenced by amino acid substitutions in the neuraminidase (NA) of A(H3N2) viruses. It is particularly useful for the characterization of viruses that either grow to low HA titer and/or undergo an abortive infection resulting in an inability to form plaques in cultured cells.

This study describes an imaging-based micro-neutralization assay to analyze the antigenic relationships between viruses. The protocol employs a flatbed scanner and has four steps, including titration, titration quantitation, neutralization, and neutralization quantitation. The assay works well with current circulating influenza A(H1N1)pdm09, A(H3N2), and B viruses.

定量微中和试验的优化

The micro-neutralization (MN) assay is a standard technique for measuring the infectivity of the influenza virus and the inhibition of virus replication. ...

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol specifically intended to measure antigen-specific killing without an infection. This protocol is designed and describes methods to overcome these limitations by allowing for the detection of antigen-specific killing of a target cell by CD8+ T cells in vivo. This is accomplished by merging a vaccination model with a traditional CFSE-labeled target killing assay. This combination allows the researcher to assess the antigen-specific CTL potential directly and quickly as the assay is not dependent upon tumor growth or infection. In addition, the readout is based on flow cytometry and so should be readily accessible to most researchers. The major limitation of the study is identifying the timeline in vivo that is appropriate to the hypothesis being tested. Variations in antigen strength and mutations in the T cells that may result in differential cytolytic function need to be carefully assessed to determine the optimal time for cell harvest and assessment. The appropriate concentration of peptide for vaccination has been optimized for hgp10025-33 and OVA257-264, but further validation would be needed for other peptides that may be more appropriate to a given study. Overall, this protocol allows a quick assessment of killing function in vivo and can be adapted to any given antigen.

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

The goal of this protocol is to allow for detection of in vivo antigen-specific killing of a target cell in a murine model.

在体内应用免疫模型检测抗原特异性 T 细胞细胞功能的试验

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol ...

确定使用血凝检测血清中的反应和滴度

摘要

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测量50％的溶血性补充（CH₅₀）活性的血清

确定临床抗体样品的吞噬活性

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