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/pubmed?term=((Determination+of+lymphocyte+division+by+flow+cytometry%5BTitle%5D)+AND+%22J.+Immunol.+Methods%22%5BJournal%5D)">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. Use of the intracellular fluorescent dye CFSE to monitor lymphocyte migration and proliferation. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aParish%2C+CR+%22Current+protocols+in+immunology%22">Current protocols in immunology</a>. Coligan, J. E. , (2009).</li>
<li>Quah, B. J., Warren, H. S., Parish, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Monitoring+lymphocyte+proliferation+in+vitro+and+in+vivo+with+the+intracellular+fluorescent+dye+carboxyfluorescein+diacetate+succinimidyl+ester%5BTitle%5D)+AND+%22Nature+Protocols%22%5BJournal%5D)">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/pubmed?term=((Fluorescent+dyes+for+lymphocyte+migration+and+proliferation+studies%5BTitle%5D)+AND+%22Immunol+Cell+Biol%22%5BJournal%5D)">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。

<table><tbody><tr><td>CFDA,SE</td><td>Molecular Probes, Life Technologies</td><td></td><td></td></tr><tr><td>DMSO</td><td>Cambridge Isotope Laboratories</td><td></td><td></td></tr><tr><td>Lymphocytes</td><td>eg mouse or human</td><td></td><td></td></tr><tr><td>RPMI</td><td>GIBCO, by Life Technologies</td><td></td><td></td></tr><tr><td>FCS</td><td>SAFC Global</td><td></td><td></td></tr><tr><td>10-15ml conical tubes</td><td>Falcon BD</td><td></td><td></td></tr><tr><td>PBS</td><td>GIBCO, by Life Technologies</td><td></td><td></td></tr><tr><td>Aluminium foil</td><td>Capral Aluminium</td><td></td><td></td></tr><tr><td>Vortex</td><td>Scientific Industries Inc.</td><td></td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>Flow cytometer</td><td>BD Biosciences</td><td></td><td></td></tr></tbody></table>

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 ...

the-use-carboxyfluorescein-diacetate-succinimidyl-ester-cfse-to

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

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

91.9K 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

Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells

Carboxyfluorescein diacetate succinimidyl ester (CFSE) can be used to easily and quickly label a cell population of interest for in vivo investigation. This labeling has classically been used to study proliferation and migration. In the method presented here, we have shortened the timeline after adoptive transfer to look at survival and killing of epitope specific CFSE labeled target cells4-6. The level of specific killing of a CD8 + T cell clone can indicate the quality of the response, as their quantity may be misleading. Specific CD8+ T cells can become functionally exhausted over time with a decline in cytokine production and killing7,8. Also, certain CD8 + T cell clones may not kill as well as others with differing TCR specificities9. For effective Cell Mediated Immunity (CMI), antigens must be identified that produce not only adequate numbers of responding T cells, but also functionally robust responding T cells. Here we assess the percent cell specific killing of two peptide specific T cell clones in BALB/c mice.

Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells

Many infections elicit a strong CTL response, but occasionally, the quantity of responding cells does not correlate to control of the pathogen1. One measure of CTL quality is their ability to kill specifically2. CFSE labeling of target cells can be used to investigate this CTL response quality in vivo3,4.

抗原特异性在体内使用CFSE标记的靶细胞的杀伤含量

Carboxyfluorescein diacetate succinimidyl ester (CFSE) can be used to easily and quickly label a cell population of interest for in vivo investigation.  ...

科研

免疫与感染

Identification and Isolation of Slow-Dividing Cells in Human Glioblastoma Using Carboxy Fluorescein Succinimidyl Ester (CFSE)

Tumor heterogeneity represents a fundamental feature supporting tumor robustness and presents a central obstacle to the development of therapeutic strategies1. To overcome the issue of tumor heterogeneity, it is essential to develop assays and tools enabling phenotypic, (epi)genetic and functional identification and characterization of tumor subpopulations that drive specific disease pathologies and represent clinically relevant targets. It is now well established that tumors exhibit distinct sub-fractions of cells with different frequencies of cell division, and that the functional criteria of being slow cycling is positively associated with tumor formation ability in several cancers including those of the brain, breast, skin and pancreas as well as leukemia2-8. The fluorescent dye carboxyfluorescein succinimidyl ester (CFSE) has been used for tracking the division frequency of cells in vitro and in vivo in blood-borne tumors and solid tumors such as glioblastoma2,7,8. The cell-permeant non-fluorescent pro-drug of CFSE is converted by intracellular esterases into a fluorescent compound, which is retained within cells by covalently binding to proteins through reaction of its succinimidyl moiety with intracellular amine groups to form stable amide bonds9. The fluorescent dye is equally distributed between daughter cells upon divisions, leading to the halving of the fluorescence intensity with every cell division. This enables tracking of cell cycle frequency up to eight to ten rounds of division10. CFSE retention capacity was used with brain tumor cells to identify and isolate a slow cycling subpopulation (top 5% dye-retaining cells) demonstrated to be enriched in cancer stem cell activity2. 
This protocol describes the technique of staining cells with CFSE and the isolation of individual populations within a culture of human glioblastoma (GBM)-derived cells possessing differing division rates using flow cytometry2. The technique has served to identify and isolate a brain tumor slow-cycling population of cells by virtue of their ability to retain the CFSE labeling.

Identification and Isolation of Slow-Dividing Cells in Human Glioblastoma Using Carboxy Fluorescein Succinimidyl Ester CFSE

This video protocol demonstrates the application of the fluorescent dye carboxyfluorescein succinimidyl ester (CFSE) for the identification and separation of different sub-populations of cells in human glioblastoma based on frequency of cell division.

慢分裂的细胞在人脑胶质瘤羧基荧光素琥珀酰亚胺酯（CFSE）的分离与鉴定

Tumor heterogeneity represents a fundamental feature supporting tumor robustness and presents a central obstacle to the development of therapeutic ...

医学

Functional Characterization of Regulatory Macrophages That Inhibit Graft-reactive Immunity

Macrophage accumulation in transplanted organs has long been recognized as a feature of allograft rejection1. Immunogenic monocytes infiltrate the allograft early after transplantation, mount a graft reactive response against the transplanted organ, and initiate organ rejection2. Recent data suggest that suppressive macrophages facilitate successful long-term transplantation3 and are required for the induction of transplantation tolerance4. This suggests a multidimensional concept of macrophage ontogeny, activation, and function, which demands a new roadmap for the isolation and analysis of macrophage function5. Due to the plasticity of macrophages, it is necessary to provide a methodology to isolate and characterize macrophages, depending on the tissue environment, and to define their functions according to different scenarios. Here, we describe a protocol for immune characterization of graft-infiltrating macrophages and the methods we used to functionally evaluate their capacity to inhibit CD8+ T proliferation and to promote CD4+Foxp3+ Treg expansion in vitro.

Macrophages are plastic cells of the hematopoietic system that have a crucial role in protective immunity and homeostasis. In this report, we describe optimized in vitro techniques to phenotypically and functionally characterize graft-infiltrating regulatory macrophages that accumulate in the transplanted organ under tolerogenic conditions.

抑制接枝反应性免疫的调节性巨噬细胞的功能表征

Macrophage accumulation in transplanted organs has long been recognized as a feature of allograft rejection1. Immunogenic monocytes infiltrate the ...

Isolation and Activation of Murine Lymphocytes

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading pathogen1,2. Understandably, such intricate abilities are controlled by a large number of molecules involved in various cellular processes to ensure timely and spatially regulated immune responses3. Here, we describe experimental procedures that allow rapid isolation of highly purified murine lymphocytes using magnetic cell sorting technology. The resulting purified lymphocytes can then be subjected to various in vitro or in vivo functional assays, such as the determination of lymphocyte signaling capacity upon stimulation by immunoblotting4 and the investigation of proliferative abilities by 3H-thymidine incorporation or carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling5-7. In addition to comparing the functional capacities of control and genetically modified lymphocytes, we can also determine the T cell stimulatory capacity of antigen-presenting cells (APCs) in vivo, as shown in our representative results using transplanted CFSE-labeled OT-I T cells.

Lymphocytes are the major players in adaptive immune responses. Here, we present a lymphocyte purification protocol to determine the physiological functions of the desired molecules in lymphocyte activation in vitro and in vivo. The described experimental procedures are suitable for comparing functional capacities between control and genetically modified lymphocytes.

分离及小鼠淋巴细胞的激活

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading ...

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 ...

Fluorescent Dye Labeling of Erythrocytes and Leukocytes for Studying the Flow Dynamics in Mouse Retinal Circulation

The retinal and choroidal blood flow dynamics may provide insight into the pathophysiology and sequelae of various ocular diseases, such as glaucoma, diabetic retinopathy, age-related macular degeneration (AMD) and other ocular inflammatory conditions. It may also help to monitor the therapeutic responses in the eye. The proper labeling of the blood cells, coupled with live-cell imaging of the labeled cells, allows for the investigation of the flow dynamics in the retinal and choroidal circulation. Here, we describe the standardized protocols of 1.5% indocyanine green (ICG) and 1% sodium fluorescein labeling of mice erythrocytes and leukocytes, respectively. Scanning laser ophthalmoscopy (SLO) was applied to visualize the labeled cells in the retinal circulation of C57BL/6J mice (wild type). Both methods demonstrated distinct fluorescently labeled cells in the mouse retinal circulation. These labeling methods can have wider applications in various ocular disease models.

Live-cell imaging of the labeled blood cells in ocular circulation can provide information about inflammation and ischemia in diabetic retinopathy and age-related macular degeneration. A protocol to label blood cells and image the labeled cells in the retinal circulation is described.

用于研究小鼠视网膜循环中流动动力学的红细胞和白细胞的荧光染料标记

The retinal and choroidal blood flow dynamics may provide insight into the pathophysiology and sequelae of various ocular diseases, such as glaucoma, ...

Quantification of Proliferating Human Antigen-specific CD4+ T Cells using Carboxyfluorescein Succinimidyl Ester

Described is a simple, in vitro, dye dilution-based method for measuring antigen-specific CD4+ T cell proliferation in human peripheral blood mononuclear cells (PBMCs). The development of stable, non-toxic, fluorescent dyes such as carboxyfluorescein succinimidyl ester (CFSE) allows for rare, antigen-specific T cells to be distinguished from bystanders by diminution in fluorescent staining, as detected by flow cytometry. This method has the following advantages over alternative approaches: (i) it is very sensitive to low-frequency T cells, (ii) no knowledge of the antigen or epitope is required, (iii) the phenotype of the responding cells can be analyzed, and (iv) viable, responding cells can be sorted and used for further analysis, such as T cell cloning.

Quantification of Proliferating Human Antigen-specific CD4+ T Cells using Carboxyfluorescein Succinimidyl Ester

Presented here is a protocol for measuring proliferating CD4+ T cells in response to antigenic proteins or peptides using dye dilution. This assay is particularly sensitive to rare antigen-specific T cells and can be modified to facilitate cloning of antigen-specific cells.

增殖人类抗原特异性CD4+ T细胞使用卡博曲莱辛苏奇尼米酯的定量

Described is a simple, in vitro, dye dilution-based method for measuring antigen-specific CD4+ T cell proliferation in human peripheral blood mononuclear ...

Rapid In Vivo Assessment of Adjuvant's Cytotoxic T Lymphocytes Generation Capabilities for Vaccine Development

The assessment of modern sub-unit vaccines reveals that the generation of neutralizing antibodies is important but not sufficient for adjuvant selection. Therefore, adjuvants with both humoral and cellular immuno-stimulatory capabilities that are able to promote cytotoxic T lymphocytes (CTL) responses are urgently needed. Thus, faithful monitoring of adjuvant candidates that induce cross-priming and subsequently enhance CTL generation represents a crucial step in vaccine development. In here we present an application for a method that uses SIINFEKL-specific (OT-I) T cells to monitor the cross-presentation of the model antigen ovalbumin (OVA) in vivo in the presence of different adjuvant candidates. This method represents a rapid test to select adjuvants with the best cross-priming capabilities. The proliferation of CD8+ T cells is the most valuable indication of cross-priming and it is also regarded as a correlate of adjuvant-induced cross-presentation. This feature can be evaluated in different immune organs like lymph nodes and spleen. The extent of the CTL generation can also be monitored, thereby giving insights on the nature of a local (draining lymph node mainly) or a systemic response (distant lymph nodes and/or spleen). This technique further allows multiple modifications for testing drugs that can inhibit specific cross-presentation pathways and also offers the possibility to be used in different strains of conventional and genetically modified mice. In summary, the application that we present here will be useful for vaccine laboratories in industry or academia that develop or modify chemical adjuvants for vaccine research and development.

Rapid In Vivo Assessment of Adjuvant's Cytotoxic T Lymphocytes Generation Capabilities for Vaccine Development

We present here an application for a standard immunological technique (CFSE stained OT-I proliferation) intended to rapidly monitor adjuvant-mediated cytotoxic T lymphocyte (CTL) generation in vivo. This fast estimation of CTL capacities is useful for the development of prophylactic vaccines against intracellular pathogens as well as therapeutic cancer vaccines.

疫苗开发中佐剂细胞毒性 T 淋巴细胞生成能力的快速体内评价

The assessment of modern sub-unit vaccines reveals that the generation of neutralizing antibodies is important but not sufficient for adjuvant selection. ...

Cell Cycle Analysis: Assessing CD4 and CD8 T Cell Proliferation After Stimulation Using CFSE Staining and Flow Cytometry

Source: Perchet Thibaut1,2,3, Meunier Sylvain1,2,3, Sophie Novault4, Rachel Golub1,2,3 
1 Unit for Lymphpoiesis, Department of Immunology, Pasteur Institute, Paris, France 
2 INSERM U1223, Paris, France 
3 Universit&#233; Paris Diderot, Sorbonne Paris Cit&#233;, Cellule Pasteur, Paris, France 
4 Flow Cytometry Platfrom, Cytometry and Biomarkers UtechS, Center for Translational Science, Pasteur Institute, Paris, France
The cell cycle is a universal process of life. During the cell cycle, a cell undergoes several modifications to divide into two daughter cells. This mechanism occurs throughout an organism&#39;s life in response to its needs. Cell divisions and embryonic development produce a full organism from a single-celled zygote. During adulthood, the cell cycle is central to many critical biological processes, such as tissue repairs.
Mechanisms of cell division are tightly controlled events where the cell undergoes stepwise modifications before final division. Cells that are not yet in the cycle are described as being in the Gap 0 (G0) phase. During this stage the cell is considered quiescent. When the cell starts to cycle, four distinct phases are recognized: Gap 1 (G1), Synthesis (S), Gap 2 (G2) and Mitosis (M). G1 phase is a checkpoint for resources needed by the cell for DNA synthesis. Then, S phase occurs, and DNA replication starts, followed by the G2 interphase, another checkpoint that controls all elements necessary to for the cell to divide. Finally, the cell enters mitosis and divides into two daughter cells.
Cell division is a highly informative parameter in many different biological systems. In the field of immunology, analysis of leukocyte proliferation can indicate the mechanism of the immune response. Other domains of investigation also rely on cell cycle analysis. For instance, analysis of the cell cycle during tumor development has improved our understanding of cancer.
Many fluorescent dyes are now available for tracking cell proliferation. These dyes differ in their chemical and spectral properties. Two different classes of dyes exist: protein dyes permanently combine with protein by forming a covalent bond, and membrane dyes stably intercalate within cell membranes via strong hydrophobic associations. In vitro and in vivo studies of immune cell proliferation by flow cytometry are among the most common applications of both classes of cell tracking dyes (1, 2).
CFSE (Carboxyfluorescein succinimidyl ester) is a fluorescent dye that marks dividing cells. Initially, all the cells receive the same amount of dye; dividing cells evenly split the dye they received between their two daughter cells. Consequently, the cell cycle can be followed by the progressive decrease of dye intensity in the cells. CFSE staining is followed by conventional multi-parametric flow cytometry, a high-throughput, fluorescence-based technology that allows phenotypic and functional characterization of cells based on their degree of CFSE staining (3).
In the following experiment, we assess the proliferation of CD4+ and CD8+ T cells in-vitro, following CD3 stimulation, using CFSE staining and flow cytometry.

Cell Cycle Analysis: Assessing CD4 and CD8 T Cell Proliferation

细胞周期分析:使用CFSE染色和流细胞测定法评估刺激后CD4和CD8 T细胞增殖

Source: Perchet Thibaut1,2,3, Meunier Sylvain1,2,3, Sophie Novault4, Rachel Golub1,2,3
1 Unit for Lymphpoiesis, Department of Immunology, Pasteur ...

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

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

一种用于评估单核细胞对白细胞增殖免疫调节影响的体外测定

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