在这篇文章中报道的工作是由国家卫生和医学研究委员会（NHMRC）澳大利亚项目资助（CRP）和一个NHMRC资助计划（CRP和BJCQ）的支持。此外，BJCQ是NHMRC彼得多尔蒂博士后研究员。

1）试剂设定 1.1）CFSE原液 CFSE染料羧基荧光素二醋酸琥珀酰亚胺酯（CFDA，SE）（兆瓦557.47克/摩尔）购买，通常在25毫克小瓶。溶于8.96 mL的DMSO为5毫米的最后原液（如50-100微升等分几个月，在-20 ° C店）25毫克。 CFDA，SE将与水溶液反应，因此它是至关重要的，这种接触是在存储过程中避免。 1.2）淋巴细胞隔离或从小鼠脾脏和淋巴结的淋巴细胞和悬浮在1毫升培养液中（如的RPMI）5％热灭活（HI）的小牛血清（FCS），与一个细胞浓度在0.5 × 10 6 - 10 × 10 7 /毫升。 2）CFSE标签 2.1）常规方法： <ol class="lalpha"><li>在中期和地点1 mL体积，在一个新的（非湿）10 mL锥形管底部，仔细，彻底重悬细胞。 </li><li>铺设管水平（使用非湿润管会阻止移动和过早与CFDA，SE的解决方案混合的1毫升细胞悬液）。 </li><li>小心加110μL的PBS以确保它不会使细胞溶液接触的管道顶部的塑料非湿部分。 </li><li>重悬于1.1 CFDA，SE 5毫米的股票在110μL的PBS液。 </li><li>快速帽管，倒置和涡获得快速均匀混合的解决方案。注意，CFSE染料在终浓度为5微米，但是，最佳浓度可能被确定为每个准备一批，然后检查每6个月，以确保它是有效的。 </li><li> 5分钟在室温下孵育细胞。注意CFDA，SE后转换为CFSE（见讨论）成为荧光染料，从而容易出现过度照射漂白。因此，最好是从这个角度来保护管，例如，用铝箔覆盖。 </li><li>洗细胞稀释在20 ° C PBS含5％您好FCS 10卷，300 XG沉积在离心5分钟，在20 ° C和丢弃上清液。重复洗两次。 </li></ol> 2.2）的替代方法，这也是适合于较高的手机号码（10 - 30 × 10 7 ）： <ol class="lalpha"><li> 1毫升PBS及快速添加这个彻底的重新悬浮细胞，以1毫升加入2μL的5毫米​​股票准备一个2 ×（10μM）CFDA，SE的解决方案，然后迅速帽管和仰拱和涡得到快速均匀混合。 </li><li>在室温下5分钟的孵育细胞和洗涤步骤2.1（F）和2.1（G）。 </li></ol> 2.3）的细胞可以被应用到的利益诱导细胞分裂的协议。可用于标记细胞在体外和体内实验。 2.4）在增殖实验完成，细胞收获和流式细胞仪分析（见下文有代表性的例子）。 3）代表性的成果为了评估淋巴细胞增殖CFSE稀释，这是需要了解的有用不可分割的细胞（即最大荧光）和非标记的细胞（即最低荧光）的荧光。因此，你的实验设计应考虑到这些控件。下面是在体外和体内实验使用CFSE流式细胞仪来衡量的CD8 + T细胞分裂的例子。 3.1） 在体外刺激图1显示的CFSE型材CFSE -标记的卵白蛋白（OVA）的特异性T细胞受体的转基因CD8 + OT - I T细胞与树突状细胞与卵子的各种款项后3天的文化。 CD8 + T细胞的纯化OT -我小鼠的脾和淋巴结肿大与CFSE和1 × 10 5细胞培养3.3 × 10 4直流标记。直流脉冲的OVA 1小时在37 ° C，洗两次前文化。经过3天，收获和抗- CD8 - APC的抗体和Hoechst 33258荧光标记，然后用流式细胞仪（收集到50,000个事件）分析细胞。演唱会（Hoechst 33258荧光 - ）CD8 +细胞显示。作为对照，CD8 +的T细胞单独培养，显示了非细胞CFSE强度，同时非标记细胞的自体荧光检测细胞分裂的细胞和限制。请注意在这个例子中，每个小组4 CFSE峰可以看出，这表明，这些细胞都发生了多达3个分支。 3.2） 在体内的刺激图2显示了CFSE型材CFSE标记的OVA特异性T细胞受体转基因CD8 + OT - I T细胞体内后3天我n为20微克OVA存在。 CD8 + T细胞的纯化CD45.1 congenic OT我小鼠的脾和淋巴结肿大，CFSE和5 × 10 6细胞注入尾静脉的C56BL/6J小鼠（CD45.2 +）第四标记。经过2小时的小鼠注射四卵子与20微克。 3天之后，从小鼠脾脏，制成单细胞悬液，抗- B220 - PerCP，抗- CD8 - APC - Cy7和反CD45.1 PE - Cy7抗体和Hoechst 33258标记，然后分析流式细胞仪（百万事件收集）。现场（33258赫斯特 - ）B220 -细胞都显示在左面板和右面板中显示的门CD8 + CD45.1 +细胞。作为对照，未刺激CFSE标记的CD8 + T细胞，显示了非细胞CFSE强度，同时非标细胞的自体荧光检测细胞分裂的细胞和限制。请注意，使用CD45的异型差异是重要的解决转让，CD8 + T细胞，从主机，因为他们是一个总CD8 + T细胞（左侧面板）未成年人的一个子集。请注意，大多数细胞属于7 CFSE峰，在这个例子中（右图），这表明，这些细胞都发生了高达6师。 <img src="/files/ftp_upload/2259/2259fig1.jpg" alt="图1" /> 图1。能力CFSE -标记的卵清蛋白（OVA）的特定T细胞受体的转基因CD8 + OT，我T 细胞在不同浓度的OVA 致敏体外到直流的回应，经过3天的文化显示的数据。注意非，划分的CD8人口+ T在缺乏抗原（浅灰色的柱状图）和非标记的人口（深灰色直方图）的自体荧光的细胞。该图描绘CD8 + T的划分基于CFSE稀释峰的1-3倍除以在较高的抗原浓度与更多的T细胞，细胞。 <img src="/files/ftp_upload/2259/2259fig2.jpg" alt="图2" /> 图2。能力CFSE -标记的卵清蛋白（OVA）的特异性T细胞受体的转基因CD8 + OT，我T细胞，时过继到C57BL / 6小鼠转移响应的OVA，数据显示继转移后3天左面板：。CD8 +，CD45的。 1 + OT - 1 T细胞在受体小鼠。右面板：CFSE扩散轮廓。注意：非人口的CD8 + T细胞在受体小鼠不接受抗原（浅灰色的柱状图）和非标记的淋巴细胞（深灰色直方图）的自体荧光。该图描绘CD8 + T细胞分为1-6倍的基础上CFSE稀释峰。 <img src="/files/ftp_upload/2259/2259fig3.jpg" alt="图3" /> 图3。一个代表各种分子事件发生在细胞与羧基荧光素二醋酸琥珀酰亚胺酯（CFDA，SE）标签的过程的详细信息。看到讨论的案文。注：通用的缩写“CFSE”，CFDA，SE，是用来描述一般的标签试剂和标签程序。图基于教区4。

<ol>
	<li>Lyons, A. B., Parish, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+lymphocyte+division+by+flow+cytometry.">Determination of lymphocyte division by flow cytometry.</a> J. Immunol. Methods. 171, 131-137 (1994).</li><li>Parish, C. R., Glidden, M. H., Quah, B. J., Warren, H. S., Coligan, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+the+intracellular+fluorescent+dye+CFSE+to+monitor+lymphocyte+migration+and+proliferation.">Use of the intracellular fluorescent dye CFSE to monitor lymphocyte migration and proliferation.</a> Current protocols in immunology. , (2009).</li><li>Quah, B. J., Warren, H. S., Parish, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+lymphocyte+proliferation+in+vitro+and+in+vivo+with+the+intracellular+fluorescent+dye+carboxyfluorescein+diacetate+succinimidyl+ester.">Monitoring lymphocyte proliferation in vitro and in vivo with the intracellular fluorescent dye carboxyfluorescein diacetate succinimidyl ester.</a> Nature Protocols. 2, 2049-2056 (2007).</li><li>Parish, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+dyes+for+lymphocyte+migration+and+proliferation+studies.">Fluorescent dyes for lymphocyte migration and proliferation studies.</a> Immunol Cell Biol. 77, 499-508 (1999).</li></ol>

为了明白如何CFSE的工作原理，以及为什么需要快速统一的标签，是其在使用扩散分析，它是有用的了解CFSE细胞标记的分子基础。这可分为两个阶段，1）进入细胞和2）蛋白标签（图3）。 1）是介导进入细胞到细胞的染料进入diacetylated形式，染料羧基荧光素二醋酸琥珀酰亚胺酯（CFDA，SE）。醋酸乙烯酯使染料的高度膜permeant允许其跨质膜的快速通量。酯酶，细胞内的存在，切割醋酸CFDA，SE，从而引起的要少得多的膜permeant染料CFSE形式，因此集中在细胞内的染料。 2）蛋白质标记：虽然都CFDA，SE和CFSE氨基反应琥珀侧链，只有CFSE荧光。 CFSE细胞内的高浓度，有利于快速和高层次的细胞内蛋白质的标签。 CFSE细胞的标签，必须迅速进行，以获得一个均匀的标记细胞的人口，这是为解决经历了几个部门，在代表性的结果（图1和2）所示的细胞至关重要。

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the use of carboxyfluorescein diacetate succinimidyl ester (cfse) to monitor lymphocyte proliferation

羧基荧光素琥珀酰亚胺酯（CFSE）是一个有效的和流行的手段来监测淋巴细胞分工 1-3 。 CFSE共价键的标签与荧光染料，羧基的长寿命的细胞内分子。因此，一个CFSE标记的细胞分裂时，其后代具有羧基荧光素标记的分子数的一半，因此每个细胞分裂，可通过测量细胞通过流式细胞仪检测荧光的相应减少评估。 CFSE的标签与特别低方差的荧光强度高，再加上其低细胞毒性淋巴细胞人口的能力，使一个理想的染料来衡量细胞分裂。由于它是一种基于荧光染料，它也与其他荧光使其适用于多色流式细胞仪的广泛兼容。本文介绍标签为目的的监控多达8个细胞分裂的小鼠淋巴细胞通常使用的程序。这些标记的细胞，可用于在体外和体内研究。

Watch this Scientific Journal Video about The Use of Carboxyfluorescein Diacetate Succinimidyl Ester CFSE to Monitor Lymphocyte Proliferation at JoVE.com

The Use of Carboxyfluorescein Diacetate Succinimidyl Ester CFSE to Monitor Lymphocyte Proliferation

CFSE共价键的标签与荧光染料，羧基的长寿命的细胞内分子。因此，一个CFSE标记的细胞分裂时，它的后代有一半的荧光量，从而可以被用来评估细胞分裂。本文介绍与CFSE标记的小鼠淋巴细胞通常使用的程序。

羧基荧光素二醋酸琥珀酰亚胺酯（CFSE）用于监视淋巴细胞增殖

Carboxyfluorescein succinimidyl ester (CFSE) is an effective and popular means to monitor lymphocyte division1-3.  CFSE covalently labels long-lived ...

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Research

JoVE Journal

Immunology and Infection

The Use of Carboxyfluorescein Diacetate Succinimidyl Ester (CFSE) to Monitor Lymphocyte Proliferation

91.3K 次观看.  John Curtin School of Medical Research, Australian National University. CFSE共价键的标签与荧光染料，羧基的长寿命的细胞内分子。因此，一个CFSE标记的细胞分裂时，它的后代有一半的荧光量，从而可以被用来评估细胞分裂。本文介绍与CFSE标记的小鼠淋巴细胞通常使用的程序。

视频: The Use of Carboxyfluorescein Diacetate Succinimidyl Ester CFSE to Monitor Lymphocyte Proliferation

Finger-stick Blood Sampling Methodology for the Determination of Exercise-induced Lymphocyte Apoptosis

Exercise is a physiological stimulus capable of inducing apoptosis in immune cells. To date, various limitations have been identified with the measurement of this phenomenon, particularly relating to the amount of time required to isolate and treat a blood sample prior to the assessment of cell death. Because of this, it is difficult to determine whether reported increases in immune cell apoptosis can be contributed to the actual effect of exercise on the system, or are a reflection of the time and processing necessary to eventually obtain this measurement. In this article we demonstrate a rapid and minimally invasive procedure for the analysis of exercise-induced lymphocyte apoptosis. Unlike other techniques, whole blood is added to an antibody panel immediately upon obtaining a sample. Following the incubation period, red blood cells are lysed and samples are ready to be analyzed. The use of a finger-stick sampling procedure reduces the volume of blood required, and minimizes the discomfort to subjects.

Exercise is capable of inducing apoptosis in immune cells. There are various measurement limitations, particularly relating to the amount of time required to isolate and treat a blood sample prior to the assessment. Demonstrated is a rapid and minimally invasive procedure for the analysis of exercise-induced lymphocyte apoptosis.

手指棒运动诱导淋巴细胞凋亡的测定血液抽样方法

Exercise is a physiological stimulus capable of inducing apoptosis in immune cells. To date, various limitations have been identified with the measurement ...

科研

免疫与感染

A Visual Assay to Monitor T6SS-mediated Bacterial Competition

Type VI secretion systems (T6SSs) are molecular nanomachines allowing Gram-negative bacteria to transport and inject proteins into a wide variety of target cells1,2. The T6SS is composed of 13 core components and displays structural similarities with the tail-tube of bacteriophages3. The phage uses a tube and a puncturing device to penetrate the cell envelope of target bacteria and inject DNA. It is proposed that the T6SS is an inverted bacteriophage device creating a specific path in the bacterial cell envelope to drive effectors and toxins to the surface. The process could be taken further and the T6SS device could perforate other cells with which the bacterium is in contact, thus injecting the effectors into these targets. The tail tube and puncturing device parts of the T6SS are made with Hcp and VgrG proteins, respectively4,5.
The versatility of the T6SS has been demonstrated through studies using various bacterial pathogens. The Vibrio cholerae T6SS can remodel the cytoskeleton of eukaryotic host cells by injecting an "evolved" VgrG carrying a C-terminal actin cross-linking domain6,7. Another striking example was recently documented using Pseudomonas aeruginosa which is able to target and kill bacteria in a T6SS-dependent manner, therefore promoting the establishment of bacteria in specific microbial niches and competitive environment8,9,10.
In the latter case, three T6SS-secreted proteins, namely Tse1, Tse2 and Tse3 have been identified as the toxins injected in the target bacteria (Figure 1). The donor cell is protected from the deleterious effect of these effectors via an anti-toxin mechanism, mediated by the Tsi1, Tsi2 and Tsi3 immunity proteins8,9,10. This antimicrobial activity can be monitored when T6SS-proficient bacteria are co-cultivated on solid surfaces in competition with other bacterial species or with T6SS-inactive bacteria of the same species8,11,12,13.
The data available emphasized a numerical approach to the bacterial competition assay, including time-consuming CFU counting that depends greatly on antibiotic makers. In the case of antibiotic resistant strains like P. aeruginosa, these methods can be inappropriate. Moreover, with the identification of about 200 different T6SS loci in more than 100 bacterial genomes14, a convenient screening tool is highly desirable. We developed an assay that is easy to use and requires standard laboratory material and reagents. The method offers a rapid and qualitative technique to monitor the T6SS-dependent bactericidal/bacteriostasis activity by using a reporter strain as a prey (in this case Escherichia coli DH5&alpha;) allowing a-complementation of the lacZ gene. Overall, this method is graphic and allows rapid identification of T6SS-related phenotypes on agar plates. This experimental protocol may be adapted to other strains or bacterial species taking into account specific conditions such as growth media, temperature or time of contact.

We describe a qualitative assay to monitor bacterial competition mediated by the Pseudomonas aeruginosa type VI secretion system (T6SS). The assay relies on the survival/killing of Escherichia coli target cells carrying a lacZ-reporter. This technique is adjustable to assess the bactericidal/bacteriostasis activity of T6SS-proficient microorganisms.

的视觉检测，以监测T6SS介导的细菌竞争

Type VI secretion systems (T6SSs) are molecular  nanomachines allowing Gram-negative bacteria to transport and inject proteins  into a wide variety of ...

Use of In vivo Imaging to Monitor the Progression of Experimental Mouse Cytomegalovirus Infection in Neonates

Human Cytomegalovirus (HCMV or HHV-5) is a life-threatening pathogen in immune-compromised individuals. Upon congenital or neonatal infection, the virus can infect and replicate in the developing brain, which may induce severe neurological damage, including deafness and mental retardation. Despite the potential severity of the symptoms, the therapeutic options are limited by the unavailability of a vaccine and the absence of a specific antiviral therapy. Furthermore, a precise description of the molecular events occurring during infection of the central nervous system (CNS) is still lacking since observations mostly derive from the autopsy of infected children. Several animal models, such as rhesus macaque CMV, have been developed and provided important insights into CMV pathogenesis in the CNS. However, despite its evolutionary proximity with humans, this model was limited by the intracranial inoculation procedure used to infect the animals and consistently induce CNS infection. Furthermore, ethical considerations have promoted the development of alternative models, among which neonatal infection of newborn mice with mouse cytomegalovirus (MCMV) has recently led to significant advances. For instance, it was reported that intraperitoneal injection of MCMV to Balb/c neonates leads to infection of neurons and glial cells in specific areas of the brain. These findings suggested that experimental inoculation of mice might recapitulate the deficits induced by HCMV infection in children. Nevertheless, a dynamic analysis of MCMV infection of neonates is difficult to perform because classical methodology requires the sacrifice of a significant number of animals at different time points to analyze the viral burden and/or immune-related parameters. To circumvent this bottleneck and to enable future investigations of rare mutant animals, we applied in vivo imaging technology to perform a time-course analysis of the viral dissemination in the brain upon peripheral injection of a recombinant MCMV expressing luciferase to C57Bl/6 neonates.

Use of In vivo Imaging to Monitor the Progression of Experimental Mouse Cytomegalovirus Infection in Neonates

Human Cytomegalovirus (HCMV) infection of neonates represents an important cause of mental retardation, yet the molecular events leading to virus-induced pathogenesis are still poorly understood. To investigate the dynamics of brain infection, we adapted whole-animal in vivo imaging to perform time-course analysis of neonates infected with a luciferase-recombinant virus.

使用<em&gt;在体内</em&gt;影像监测进展的实验小鼠巨细胞病毒感染的新生儿

Human Cytomegalovirus (HCMV or HHV-5) is a life-threatening  pathogen in immune-compromised individuals. Upon congenital or neonatal  infection, the virus ...

Using Fluorescent Proteins to Monitor Glycosome Dynamics in the African Trypanosome

Trypanosoma brucei is a kinetoplastid parasite that causes human African trypanosomiasis (HAT), or sleeping sickness, and a wasting disease, nagana, in cattle1. The parasite alternates between the bloodstream of the mammalian host and the tsetse fly vector. The composition of many cellular organelles changes in response to these different extracellular conditions2-5.
Glycosomes are highly specialized peroxisomes in which many of the enzymes involved in glycolysis are compartmentalized. Glycosome composition changes in a developmental and environmentally regulated manner4-11. Currently, the most common techniques used to study glycosome dynamics are electron and fluorescence microscopy; techniques that are expensive, time and labor intensive, and not easily adapted to high throughput analyses.
To overcome these limitations, a fluorescent-glycosome reporter system in which enhanced yellow fluorescent protein (eYFP) is fused to a peroxisome targeting sequence (PTS2), which directs the fusion protein to glycosomes12, has been established. Upon import of the PTS2eYFP fusion protein, glycosomes become fluorescent. Organelle degradation and recycling results in the loss of fluorescence that can be measured by flow cytometry. Large numbers of cells (5,000 cells/sec) can be analyzed in real-time without extensive sample preparation such as fixation and mounting. This method offers a rapid way of detecting changes in organelle composition in response to fluctuating environmental conditions.

Glycosome dynamics in African trypanosomes are difficult to study by traditional cell biology techniques such as electron and fluorescence microscopy. As a means of observing dynamic organelle behavior, a fluorescent-organelle reporter system has been used in conjunction with flow cytometry to monitor real-time glycosome dynamics in live parasites.

利用荧光蛋白监视非洲锥虫Glycosome动态

Trypanosoma brucei is a kinetoplastid parasite that causes human African trypanosomiasis (HAT), or sleeping sickness, and a wasting disease, nagana, in ...

Immunofluorescence to Monitor the Cellular Uptake of Human Lactoferrin and its Associated Antiviral Activity Against the Hepatitis C Virus

Immunofluorescence is a laboratory technique commonly used to study many aspects of biology. It is typically used to visualize the distribution and/or localization of a target molecule in cells and tissues. Immunofluorescence relies on the specificity of fluorescent-labelled antibodies against their corresponding antigens within a cell. Both direct and indirect immunofluorescence approaches can be used which rely on the use of antibodies linked with a fluorochrome. Direct immunofluorescence is less frequently used because it provides lower signal, involves higher cost and less flexibility. In contrast, indirect immunofluorescence is more commonly used because of its high sensitivity and provides an amplified signal since more than one secondary antibody can attach to each primary antibody. In this manuscript, both epifluorescence microscopy and confocal microscopy were used to monitor the internalization of human lactoferrin, an important component of the immune system, into hepatic cells. Moreover, we monitored the inhibitory potential of hLF on the intracellular replication of the Hepatitis C virus using immunofluorescence. Both the advantages and disadvantages associated with these approaches are discussed.

The human lactoferrin (hLF) is a component of the immune system. In this study, immunofluorescence assays are used to demonstrate both the hepatocellular uptake of hLF and a qualitative reduction in the hepatitis C virus replication upon treatment with hLF.

免疫监视人乳铁蛋白的细胞摄取及其相关抗病毒活性抗丙型肝炎病毒

Immunofluorescence is a laboratory technique commonly used to study many aspects of biology. It is typically used to visualize the distribution and/or ...

The Use of Flow Cytometry to Assess the State of Chromatin in T Cells

During a proper immune response, quiescent T cells become activated upon antigen presentation to their antigen-specific T cell receptor. This leads to clonal proliferation of only those T cells that bear a receptor that recognizes the antigen. Chromatin decondensation is a hallmark of T cell activation and is required for T cells to acquire the ability to proliferate after antigen engagement. This change in chromatin condensation can be detected using antibodies raised against histone proteins. These antibodies cannot bind to their epitopes in na&#239;ve T cells as well as they can in activated T cells. We describe how to simultaneously stain T cell-specific surface markers, track viability with a fixable dead cell stain, and measure chromatin status via intracellular staining of Histone H3 proteins. Stained cells are analyzed by flow cytometry and chromatin condensation status is measured as the mean fluorescence intensity (MFI) of the Histone H3 stain. Chromatin decondensation during T cell activation is demonstrated as an increase in the MFI

Flow cytometry can be utilized to assess the state of chromatin within T cells. This protocol allows scientists to interpret evidence of chromatin decondensation during T cell activation demonstrated by an increase in the mean florescence intensity (MFI) of fluorescent Histone H3 antibodies

流式细胞仪用来评估染色质的T细胞国家

During a proper immune response, quiescent T cells become activated upon antigen presentation to their antigen-specific T cell receptor. This leads to ...

Assay of Adhesion Under Shear Stress for the Study of T Lymphocyte-Adhesion Molecule Interactions

Overall, T cell adhesion is a critical component of function, contributing to the distinct processes of cellular recruitment to sites of inflammation and interaction with antigen presenting cells (APC) in the formation of immunological synapses. These two contexts of T cell adhesion differ in that T cell-APC interactions can be considered static, while T cell-blood vessel interactions are challenged by the shear stress generated by circulation itself. T cell-APC interactions are classified as static in that the two cellular partners are static relative to each other. Usually, this interaction occurs within the lymph nodes. As a T cell interacts with the blood vessel wall, the cells arrest and must resist the generated shear stress.1,2 These differences highlight the need to better understand static adhesion and adhesion under flow conditions as two distinct regulatory processes. The regulation of T cell adhesion can be most succinctly described as controlling the affinity state of integrin molecules expressed on the cell surface, and thereby regulating the interaction of integrins with the adhesion molecule ligands expressed on the surface of the interacting cell. Our current understanding of the regulation of integrin affinity states comes from often simplistic in vitro model systems. The assay of adhesion using flow conditions described here allows for the visualization and accurate quantification of T cell-epithelial cell interactions in real time following a stimulus. An adhesion under flow assay can be applied to studies of adhesion signaling within T cells following treatment with inhibitory or stimulatory substances. Additionally, this assay can be expanded beyond T cell signaling to any adhesive leukocyte population and any integrin-adhesion molecule pair.

This flow adhesion assay provides a simple, high impact model of T cell-epithelial cell interactions. A syringe pump is used to generate shear stress, and confocal microscopy captures images for quantification. The goal of these studies is to effectively quantify T cell adhesion using flow conditions.

在剪切粘结力的测定T淋巴细胞粘附分子相互作用的研究

Overall, T cell adhesion is a critical component of function, contributing to the distinct processes of cellular recruitment to sites of inflammation and ...

Detection of Trypanosoma brucei Variant Surface Glycoprotein Switching by Magnetic Activated Cell Sorting and Flow Cytometry

Trypanosoma brucei, a protozoan parasite that causes both Human and Animal African Trypanosomiasis (known as sleeping sickness and nagana, respectively) cycles between a tsetse vector and a mammalian host. It evades the mammalian host immune system by periodically switching the dense, variant surface glycoprotein (VSG) that covers its surface. The detection of antigenic variation in Trypanosoma brucei can be both cumbersome and labor intensive. Here, we present a method for quantifying the number of parasites that have 'switched' to express a new VSG in a given population. The parasites are first stained with an antibody against the starting VSG, and then stained with a secondary antibody attached to a magnetic bead. Parasites expressing the starting VSG are then separated from the rest of the population by running the parasites over a column attached to a magnet. Parasites expressing the dominant, starting VSG are retained on the column, while the flow-through contains parasites that express a new VSG as well as some contaminants expressing the starting VSG. This flow-through population is stained again with a fluorescently labeled antibody against the starting VSG to label contaminants, and propidium iodide (PI), which labels dead cells. A known number of absolute counting beads that are visible by flow cytometry are added to the flow-through population. The ratio of beads to number of cells collected can then be used to extrapolate the number of cells in the entire sample. Flow cytometry is used to quantify the population of switchers by counting the number of PI negative cells that do not stain positively for the starting, dominant VSG. The proportion of switchers in the population can then be calculated using the flow cytometry data.

Detection of Trypanosoma brucei Variant Surface Glycoprotein Switching by Magnetic Activated Cell Sorting and Flow Cytometry

African trypanosomes grown in vitro undergo antigenic variation at a low rate, such that populations are made up of parasites expressing a dominant variant surface glycoprotein (VSG) type and a small population of "switched" variants. This protocol describes a fast method for detecting and quantifying these populations.

检测<em&gt;布氏锥虫</em&gt;变体表面糖蛋白通过磁性细胞分选和流式细胞仪开关

Trypanosoma brucei, a protozoan parasite that causes both Human and Animal African Trypanosomiasis (known as sleeping sickness and nagana, respectively) ...

Development of a Hepatitis B Virus Reporter System to Monitor the Early Stages of the Replication Cycle

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), that carry foreign genes such as a reporter or marker protein in their genomes. These recombinant viruses usually faithfully mimic the life cycle of the original virus in infected cells and exhibit the same host range dependence. The development of a recombinant virus enables the efficient screening of inhibitors and the identification of specific host factors. However, to date the construction of recombinant hepatitis B virus (HBV) has been difficult because of various experimental limitations. The main limitation is the compact genome size of HBV, and a fairly strict genome size that does not exceed 1.3 genome sizes, that must be packaged into virions. Thus, the size of a foreign gene to be inserted should be smaller than 0.4 kb if no deletion of the genome DNA is to be performed. Therefore, to overcome this size limitation, the deletion of some HBV DNA is required. Here, we report the construction of recombinant HBV encoding a reporter gene to monitor the early stage of the HBV replication cycle by replacing part of the HBV core-coding region with the reporter gene by deleting part of the HBV pol coding region. Detection of recombinant HBV infection, monitored by the reporter activity, was highly sensitive and less expensive than detection using the currently available conventional methods to evaluate HBV infection. This system will be useful for a number of applications including high-throughput screening for the identification of anti-HBV inhibitors, host factors and virus-susceptible cells.

Here we describe a newly developed hepatitis B virus (HBV) reporter system to monitor the early stages of the HBV life cycle. This simplified in vitro system will aid in the screening of anti-HBV agents using a high-throughput strategy.

乙型肝炎病毒记者系统的开发监控复制周期的早期阶段

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), ...

Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining the role of the gene of interest in the development, differentiation, and function of NK cells. Recent advances related to chimeric antigen receptors (CARs) in cancer immunotherapy accentuate the need for an efficient method to deliver exogenous genes to effector lymphocytes. The efficiencies of lentiviral-mediated gene transductions into primary human or mouse NK cells remain significantly low, which is a major limiting factor. Recent advances using cationic polymers, such as polybrene, show an improved gene transduction efficiency in T cells. However, these products failed to improve the transduction efficiencies of NK cells. This work shows that dextran, a branched glucan polysaccharide, significantly improves the transduction efficiency of human and mouse primary NK cells. This highly reproducible transduction methodology provides a competent tool for transducing human primary NK cells, which can vastly improve clinical gene delivery applications and thus NK cell-based cancer immunotherapy.

The goal of this study was to formulate technologies that allow for successful gene transduction in primary natural killer (NK) cells. The dextran-mediated lentiviral transduction of human or mouse primary NK cells results in higher gene expression efficiencies. This method of gene transduction will vastly improve NK cell genetic manipulation.

葡聚糖增强小鼠和人原发性 NK 细胞的慢转导效率

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining ...