The authors would like to thank Joshua Kimani, clinical director of the research program at the University of Nairobi, for his assistance with mucosal immunology studies related to this protocol. The authors would like to acknowledge funding from CHVI grant MOP 86721.

伦理声明:曼尼托巴大学及内罗毕肯雅塔的国家医院/大学研究伦理委员会批准了这项研究，并签署知情同意从所有研究参与者获得。 媒体1。准备和CMC收集管<ol><li>制备磷酸盐缓冲盐水（PBS）溶液（137.93 mM氯化钠，2.67毫米氯化钾，8.1毫米的Na 2 HPO 4，1.47 mM的KH 2 PO 4）。高压灭菌无菌。这可以被存储在4℃几个月。 </li><li>预等分5毫升PBS入50ml Falcon管（每个样品1被收集），并在4℃储存直至样品收集时间。 </li><li>标签每个样品2-4冻存管或1.5 ml离心管中，以收集灌洗液进行存储。 </li><li>制备细胞培养基:RPMI 1640 +1％青霉素/链霉素/两性霉素B（终浓度为100单位/毫升，100微克/毫升和250纳克/毫升，respectiv伊利，没有FC / FB的补充。 </li></ol>细胞刷样品2。收藏收集细胞刷样本是必须由一个受过训练的医师或妇科医生进行非侵入性的过程。 <ol><li>同时使用细胞刷和一个木制或塑料刮板收集宫颈单个核细胞（CMC）。通过将刮刀与周围的宫颈内口的旋转（ 图1）收集的CMC从下窥器检查参与者。将细胞刷入颈管口，旋转360°，并立即放置两个细胞刷和刮具入含有5ml的PBS 50ml的falcon管。 </li><li>保持样品在冰/ 4℃，直到处理。为了保持细胞活力，处理样品在2小时内收集的。 </li><li>如果可能的话，收集绿色的顶部肝素真空采血管的外周血单个核细胞（PBMC）中分离，作为一个比较器/控制CMC分析匹配的血液样本。 </LI&gt; </ol>中医诊所3。隔离在生物安全二级实验室进行样品处理，在无菌生物安全柜，双层手套。 <ol><li>在收集，中央军委样本可能被污染的血液。排除具有从研究可见血液污染的样品，以防止混淆列入PBMC的最终样品中。由于淋巴细胞管委会样本中分离出的低号，次血液污染可以很容易地压倒管委会淋巴细胞组成部分。 </li><li>涡猎鹰管含有的细胞刷及刮刀45秒，从刷游离细胞。在此过程中样品可能成为泡沫状;这是正常的。 </li><li>使用细胞刷刮任何遗留物质掉刮板，并丢弃刮成漂白剂溶液。收集剩余附着在刷/刮刀的任何单元格，使用新鲜的手套挤拇指和食指之间的刮刀。滑下刷，使细胞和PBS，并收集在同一个50ml管中。丢弃细胞刷成漂白剂溶液，并丢弃的手套。 重要提示:使用一个新的手套对每个样品。 </li><li>适合100微米尼龙细胞过滤到新的50ml falcon管。使用移液管，收集从样品管中的PBS-细胞悬浮液，并通过过滤器到干净试管中进行过滤。 </li><li>加入5 ml的RPMI 1640中，以原始样品管中。使用移液管，洗样品管的侧面与RPMI，并通过过滤器转移到新的收集管。洗尼龙过滤器的RPMI的底部为好。 </li><li>离心样品10分钟，在514×g下。关键的是要在该步骤中离开离心机制动切断，以防止在CMC粒料撞出。 </li><li>轻轻地从管取出上清液，注意不要打扰颗粒。颗粒大小会变化很大;在这样我的样品，将沉淀是由于大量上皮细胞的存在相当大的，而在其他情况下，将丸粒是勉强可见和高透明度。 </li><li>由猎鹰管轻轻搅动悬浮颗粒。加入5毫升的PBS洗涤样品。 </li><li>再次离心在514 XG没有离心制动器10分钟。 </li><li>小心弃去上清液并重新悬浮沉淀如上述。这些细胞是准备或者冷冻保存（使用PBMC冷冻保存协议），刺激或流式细胞仪分型。 </li></ol> 4，CMC表面染色和流式细胞仪<ol><li>从在100μl封闭液的步骤3.10重悬沉淀并转移要么到96孔板或FACS管。封闭液（方框Fcγ受体）的配方如下:1.8微升小鼠IgG（最终浓度0.2微克/微升），5微升的FBS和93.2微升FACS洗涤液（PBS +2％FBS）中。染色可在96孔板中进行，如果粒料的大小是足够小的，但可能需要在FACS管中进行，如果颗粒是常规大。它相应滴定抗体是很重要的。 </li><li>阻断样品在冰上/ 10分钟，在4℃下</li><li>在96孔板中加入100μlFACS洗涤液（PBS +2％FBS）中，或加入500μl在FACS管洗​​涤细胞。离心机在600×g下在4℃下10分钟，使用于低制动（或关闭，如果低速制动器设置是不可用的）。 </li><li>除去上清液和重悬细胞沉淀通过搅拌。添加活力染色（现场死可以解决的可行性染料）。根据制造商的说明制备的可行性染料。它可能需要被滴定为在每个实验/流式细胞仪的设置使用。孵育在黑暗中30分钟，在4℃下</li><li>洗涤细胞100μl/500微升PBS（对于96孔板/ FACS管）。离心600×g离心10分钟，低/无刹车。删除supernatan吨，重悬细胞。 </li><li>孵育细胞表面标记的抗体的混合物，在100微升的总体积。重要提示:优化和滴定样品染色前各流式细胞仪面板。孵育细胞在黑暗中30分钟，在4℃下</li><li>重复步骤4.5。如果染色在96 - 孔板中，细胞转移到FACS管中，并稀释至750微升FACS洗涤的含1％多聚甲醛（PFA）的最终体积（或CytoFix，稀释1中4）。进行数据采集的流式细胞仪。 </li></ol> 5，收集和宫颈阴道灌洗的制备（CVL） <ol><li>此前细胞刷取样，用注射器2毫升无菌PBS冲洗宫颈内口和阴道后穹窿吸出灌洗。 </li><li>吸气式灌洗样品收集到15毫升猎鹰管并保持在冰上/ 4℃，直到处理。 </li><li>在400×g离心7分钟，以除去细胞碎片离心样品。 </li><li>合作llect上清液，等分试样的样品为1毫升或500微升的等分试样，并储存于-70℃。等分可以解冻ELISA或CVL蛋白微球阵列分析，但要避免多次冻结/解冻周期。 </li><li>如果需要的话，重悬细胞碎片的沉淀物中的RNA后来由以下制造商的方案来测量RNA表达。 </li></ol> 6，数据采集<ol><li>准备一套补偿管的抗体面板相匹配。运行补偿管调节光谱重叠。运行样品上的任何多色流式细胞仪被配置在面板中使用的荧光标记抗体。 </li><li>首先获取PBMC样本调整前向散射（FSC）和侧向散射光（SSC）电压，以检测淋巴细胞群。尽量让他们在100上每个轴的线性刻度。运行PBMC控制将使显著更容易找到管委会淋巴细胞群。 </li><li>对于CMC样品FSC的门槛可能需要相对增加至因涂抹了SSC轴在低FSC值PBMC样品。 </li><li>收购之前，每个涡管。获得整个管最大化收集淋巴细胞门事件的数目。监控机器小心，以免堵塞。 </li><li>将数据导出到流式细胞仪分析程序，如FlowJo。 </li></ol> 7，门控策略<ol><li>选择正向侧向散射高（FSC-H）/正向侧向散射面积（FSC-A）来确定单人口。 </li><li>选择时间与荧光标记（如例如FITC）来控制收购质量。变化的流量可能引入的数据失真。排除显示差异流速的任何区域。 </li><li>标识上SSC-A/FSC-A情节淋巴细胞群。用PBMC控制，以方便识别。需要注意的是管委会淋巴细胞群可能不一样清晰的PBMC控制。&lt;/ LI&gt; <li>门上SSC-A/viability染料排除死细胞与活细胞。死细胞可以非特异性结合的抗体，其中将介绍文物。 </li><li>门上的SSC-A/CD3识别的T细胞群体。从这个门，确定CD4 +和CD8 +的人群。 </li></ol>

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The authors have nothing to disclose.

面对庞大的知识差距的相对免疫力在女性生殖道（FGT），中医诊所的表型分析可以提供见解的各种各样成多个淋巴细胞群在子宫颈​​。再加上蛋白质组分析及在宫颈灌洗病毒载量的测量，抗干扰性性传播感染（STI）s和其他病原体可在解剖不同人群。 技术因素-中医诊所:隔离和CMC样品成功染色是具有挑战性的。在CMC收集协议的优化强调第一收集CVL的有效性，随?...

大多数新感染艾滋病毒引起全世界通过异性性接触传染，女性占新发感染的47％，2011年（联合国艾滋病规划署1）。了解女性生殖道（FGT），主入口门户为HIV和其他性传染病原体之一，是的路径，寻找有效的策略，以防止感染的高度重视。在生殖器粘膜免疫应答是独特的清楚和从那些在外周血2测得不同。然而，在FGT免疫动力学的当前的知识是在最好的限定。迄今为止，黏膜免疫环境的研究主要集中在肠道相关淋巴组织（GALT），它已经很清楚，感染后粘膜组织中的早期事件，对后续的疾病进展3,4的强烈冲击。从生殖器粘膜采集样本代表一个巨大的挑战，并至少部分负责LACk的FGT的免疫学的理解。解决在不同的环境，是FGT就必须有效的方法从该地区采集和分析样品的情况下主机和病原体之间的免疫动态的难题。 在FGT被分成两部分:上部生殖道，包括输卵管，子宫内膜和宫颈管，下部尿路含有宫颈和阴道（由Kaushic评论等 5）。现在还不清楚是什么，这些不同的网站的相对贡献是艾滋病毒感染，但据信，这两个网站可能导致艾滋病毒进入6。 T细胞代表在上下生殖道的白细胞的40-50％，而巨噬细胞含有约10％（以罗德里格斯加西亚评论等 2）。 T细胞可以在阴道，子宫颈，子宫内膜和被检测到。巨噬细胞是更强烈的局部性ZED的子宫内膜和肌层结缔组织比子宫颈，虽然它们可以同时在组织中被检测到。最后，浆细胞样树突细胞（PDC）和朗格汉斯细胞也可以在FGT组织中检测到。表型和免疫人群及其易于感染艾滋病毒的比例可根据荷尔蒙周期变化重要的是，使用荷尔蒙避孕药，细菌性阴道炎，或性活动5,7-9的。 多种方法已经被开发，研究免疫人群的FGT和环境。宫颈活检，宫颈cytobrushes和宫颈阴道灌洗液（CVL）10-12是整个文献中最常用的。 CVL收集由PBS灌洗是最简单的方法，并允许免疫调节蛋白，但结果在极低的细胞产量的研究中，并因此不适合用于研究FGT 13的免疫细胞群。 CVL样品，另一方面，VERY用于通过测量用的方法，如ELISA，细胞因子微球阵列14或质谱15,16的各种细胞因子，趋化因子或抗微生物因子的表达评估FGT的免疫环境是有用的。免疫细胞频率，表型和功能特性可通过宫颈细胞刷或宫颈活检取样采集子宫颈单个核细胞（CMC）来实现。 宫颈活检取样是一种侵入性方法，其增加出血的不适和风险，需要2至11天治愈以下的步骤取决于女人12的免疫状态。另一方面，宫颈cytobrushes，尽管收集的细胞的产量较低，是一种微创的，更方便的方法来收集免疫细胞从FGT。这两种方法都可以达到的CD45 +白细胞中相同的产率，但是两个连续的子cytobrushes是必要的，得到的细胞相同数量的载一n一个活检13。然而，细胞刷取样仍然为进一步体外表型通过流式细胞仪14提供了细胞的可接受的数量（大约5,000的CD45 +细胞/细胞刷）。此外，功能特性，可以进行对这些样品，如刺激和细胞内流式细胞术定量PCR或已使用细胞刷衍生的CMC识别HIV特异性免疫应答17或Th细胞极化18执行。扩张性T细胞的人口也可方便与中医诊所19功能研究。 要注意的是活检和cytobrushes采样FGT不同的部分是很重要的。活检是从宫颈12,13的上皮和基质的优越部导出，而颈cytobrushes采样宫颈内口，收集从宫颈管和推测的转化区的上皮来源的细胞。因此，细胞刷样品品尝重新只园组成的柱状上皮单层的，而活检，包括由鳞状复层上皮衬里5的区域。其结果是，通过宫颈活检和细胞刷收集的白细胞种群的性质不同而不同。活检收集CD3 + T细胞的比例较高，而cytobrushes导致收集的CD14 +单核细胞/巨噬细胞13的比例较高。 研究FGT的免疫学一直是关注了很多年20-22，我们已经积累了大量的专业知识与细胞刷源性间中医诊所的研究。我们的研究重点主要在艾滋病病毒感染，感染和HIV阴性曝光（HESN）女性性工作者来自肯尼亚内罗毕的研究。优先病毒复制在活化的T细胞23和较低的数字活化细胞，可以由艾滋病病毒在FGT有针对性有助于防止感染HIV。根据这一假设，几个STUDI线ES形容HESN性工作者谁是高度暴露于艾滋病毒在较低的免疫激活但仍然未感染的24,25，和这个静态的表型是在FGT 14还观察到。在这里，我们描述的方法进行处理，并通过评估体外流式细胞仪来源于宫颈cytobrushes CMC样品中的T细胞活化。

<table><tbody><tr><td>100 &amp;mu;m cell strainer for 50 ml Falcon tube</td><td>BD</td><td>352360</td><td>CMC processing</td></tr><tr><td>RPMI 1640</td><td>Hyclone</td><td>SH30027.01</td><td>CMC processing</td></tr><tr><td>Fetal bovine serum</td><td>Life technology</td><td>16000044</td><td>CMC processing</td></tr><tr><td>Fungizone</td><td>Life technology</td><td>15290-018</td><td>CMC processing</td></tr><tr><td>Penicillin/streptomycin</td><td>Sigma</td><td>P4333-20ml</td><td>CMC processing</td></tr><tr><td>50 ml Falcon tube</td><td>Fisher</td><td>14-959-49A</td><td>CMC processing</td></tr><tr><td>Blood Bank disposable transfer pipette</td><td>Fisher</td><td>13-711-6M</td><td>CMC processing</td></tr><tr><td>Cytobrush plus</td><td>Cooper surgical</td><td>C0121</td><td>CMC sampling</td></tr><tr><td>Disposable cervical scraper</td><td>Quick medical</td><td>2183</td><td>CMC sampling</td></tr><tr><td>15 ml Falcon tube</td><td>Fisher</td><td>14-959-70c</td><td>CVL processsing</td></tr><tr><td>1.5 ml tube Eppendorf</td><td>Fisher</td><td>05-402-18</td><td>CVL storage</td></tr><tr><td>LIVE/DEAD Fixable Cell Stain Kit</td><td>Invitrogen</td><td>Various</td><td>Flow cytometry reagent</td></tr><tr><td>Fixation buffer (4% PFA)</td><td>BD</td><td>554655</td><td>Flow cytometry regeant</td></tr><tr><td>IgG mouse</td><td>Sigma</td><td>I8765</td><td>Flow cytometry regeant</td></tr></tbody></table>

collection, isolation, and flow cytometric analysis of human endocervical samples

Despite the public health importance of mucosal pathogens (including HIV), relatively little is known about mucosal immunity, particularly at the female genital tract (FGT). Because heterosexual transmission now represents the dominant mechanism of HIV transmission, and given the continual spread of sexually transmitted infections (STIs), it is critical to understand the interplay between host and pathogen at the genital mucosa. The substantial gaps in knowledge around FGT immunity are partially due to the difficulty in successfully collecting and processing mucosal samples. In order to facilitate studies with sufficient sample size, collection techniques must be minimally invasive and efficient. To this end, a protocol for the collection of cervical cytobrush samples and subsequent isolation of cervical mononuclear cells (CMC) has been optimized. Using ex vivo flow cytometry-based immunophenotyping, it is possible to accurately and reliably quantify CMC lymphocyte/monocyte population frequencies and phenotypes. This technique can be coupled with the collection of cervical-vaginal lavage (CVL), which contains soluble immune mediators including cytokines, chemokines and anti-proteases, all of which can be used to determine the anti- or pro-inflammatory environment in the vagina.

多参数流式细胞仪是一种功能强大的工具来剖析细胞亚群的表型和功能在以前未知的组织。 CMC样品的分析可以产生两个淋巴细胞和单核细胞的人口信息与相应的门控策略。 一位代表中央军委门控策略，相比匹配的PBMC轮廓， 如图2，FSC-A与FSC-H的情节使得细胞双峰，这是非常普遍的CMC样品相比，外周血单个核细胞的排斥，甚至是过滤后（ 图2A）。质量?...

Watch this Scientific Journal Video about Collection, Isolation, and Flow Cytometric Analysis of Human Endocervical Samples at JoVE.com

Collection, Isolation, and Flow Cytometric Analysis of Human Endocervical Samples

The use of cytobrush sampling to collect lymphocytes and monocytes from the endocervix is a minimally invasive technique that provides samples for analysis of female genital tract immunity. In this protocol, we describe the collection of cytobrush samples and immune cell isolation for flow cytometry assays.

收集，分离，和人类宫颈样品的流式细胞仪分析

Despite the public health importance of mucosal pathogens (including HIV), relatively little is known about mucosal immunity, particularly at the female ...

collection-isolation-flow-cytometric-analysis-human-endocervical

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

19.0K 次观看.  University of Manitoba. The use of cytobrush sampling to collect lymphocytes and monocytes from the endocervix is a minimally invasive technique that provides samples for analysis of female genital tract immunity. In this protocol, we describe the collection of cytobrush samples and immune cell isolation for flow cytometry assays. 

视频: Collection, Isolation, and Flow Cytometric Analysis of Human Endocervical Samples

Isolation of Human Endothelial Cells from Normal Colon and Colorectal Carcinoma - An Improved Protocol

Primary cells isolated from human carcinomas are valuable tools to identify pathogenic mechanisms contributing to disease development and progression. In particular, endothelial cells (EC) constituting the inner surface of vessels, directly participate in oxygen delivery, nutrient supply, and removal of waste products to and from tumors, and are thereby prominently involved in the constitution of the tumor microenvironment (TME). Tumor endothelial cells (TECs) can be used as cellular biosensors of the intratumoral microenvironment established by communication between tumor and stromal cells. TECs also serve as targets of therapy. Accordingly, in culture these cells allow studies on mechanisms of response or resistance to anti-angiogenic treatment. Recently, it was found that TECs isolated from human colorectal carcinoma (CRC) exhibit memory-like effects based on the specific TME they were derived from. Moreover, these TECs actively contribute to the establishment of a specific TME by the secretion of different factors. For example, TECs in a prognostically favorable Th1-TME secrete the anti-angiogenic tumor-suppressive factor secreted protein, acidic and rich in cysteine-like 1 (SPARCL1). SPARCL1 regulates vessel homeostasis and inhibits tumor cell proliferation and migration. Hence, cultures of pure, viable TECs isolated from human solid tumors are a valuable tool for functional studies on the role of the vascular system in tumorigenesis. Here, a new up-to-date protocol for the isolation of primary EC from the normal colon as well as CRC is described. The technique is based on mechanical and enzymatic tissue digestion, immunolabeling, and fluorescence activated cell sorting (FACS)-sorting of triple-positive cells (CD31, VE-cadherin, CD105). With this protocol, viable TEC or normal endothelial cell (NEC) cultures could be isolated from colon tissues with a success rate of 62.12% when subjected to FACS-sorting (41 pure EC cultures from 66 tissue samples). Accordingly, this protocol provides a robust approach to isolate human EC cultures from normal colon and CRC.

Tumor endothelial cells are important determinants of the tumor microenvironment and the course of the disease. Here, a protocol for the isolation of pure and viable endothelial cells from human colorectal carcinoma and normal colon to be used in drug testing and pathogenesis research is described.

正常结肠和结直肠癌患者内皮细胞的分离--一种改进的协议

Primary cells isolated from human carcinomas are valuable tools to identify pathogenic mechanisms contributing to disease development and progression. In ...

科研

癌症研究

Automatic Separation and Collection of Cancer-Related Substances from Clinical Samples

Recently, liquid biopsies have been used to diagnose various diseases, including cancer. Body fluids contain many substances, including cells, proteins, and nucleic acids originating from normal tissues, but some of these substances also originate from the diseased area. The investigation and analysis of these substances in the body fluids play a pivotal role in the diagnosis of various diseases. Therefore, it is important to accurately separate the required substances, and several techniques are developed to be used for this purpose.
We have developed a lab-on-a-disc type of device and platform named CD-PRIME. This device is automated and has good results for sample contamination and sample stability. Moreover, it has advantages of a good acquisition yield, a short operation time, and high reproducibility. In addition, depending on the type of disc to be mounted, plasma containing cell-free DNA, circulating tumor cells, peripheral blood mononuclear cells, or buffy coats can be separated. Thus, the acquisition of a variety of materials present in the body fluids can be done for a variety of downstream applications, including the study of omics.

This paper describes the application of automated equipment to easily and efficiently separate and collect substances, such as cell-free DNA and circulating tumor cells, from whole blood.

从临床样本中自动分离和收集癌症相关物质

Recently, liquid biopsies have been used to diagnose various diseases, including cancer. Body fluids contain many substances, including cells, proteins, ...

Two Methods for Establishing Primary Human Endometrial Stromal Cells from Hysterectomy Specimens

Many efforts have been devoted to establish in vitro cell culture systems. These systems are designed to model a vast number of in vivo processes. Cell culture systems arising from human endometrial samples are no exception. Applications range from normal cyclic physiological processes to endometrial pathologies such as gynecological cancers, infectious diseases, and reproductive deficiencies. Here, we provide two methods for establishing primary endometrial stromal cells from surgically resected endometrial hysterectomy specimens. The first method is referred to as &#8220;the scraping method&#8221; and incorporates mechanical scraping using surgical or razor blades whereas the second method is termed &#8220;the trypsin method.&#8221; This latter method uses the enzymatic activity of trypsin to promote the separation of cells and primary cell outgrowth. We illustrate step-by-step methodology through digital images and microscopy. We also provide examples for validating endometrial stromal cell lines via quantitative real time polymerase chain reactions (qPCR) and immunofluorescence (IF).

Establishing primary endometrial stromal cell culture systems from hysterectomy specimens is a valuable biological technique and a crucial step prior to pursuing a vast array of research aims. Here, we describe two methods used to establish stromal cultures from surgically resected endometrial tissues of human patients.&#160;

两种方法从子宫切除标本小学建立人子宫内膜基质细胞

Many efforts have been devoted to establish in vitro cell culture systems. These systems are designed to model a vast number of in vivo processes. Cell ...

医学

Isolation of Human Lymphatic Endothelial Cells by Multi-parameter Fluorescence-activated Cell Sorting

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity but little is known about their biology. Isolating highly pure human lymphatic endothelial cells (LECs) from diseased and healthy tissue would facilitate studies of the lymphatic endothelium at genetic, molecular and cellular levels. It is anticipated that these investigations may reveal targets for new therapies that may change the clinical management of these conditions. A protocol describing the isolation of human foreskin LECs and lymphatic malformation lymphatic endothelial cells (LM LECs) is presented. To obtain a single cell suspension tissue was minced and enzymatically treated using dispase II and collagenase II. The resulting single cell suspension was then labelled with antibodies to cluster of differentiation (CD) markers CD34, CD31, Vascular Endothelial Growth Factor-3 (VEGFR-3) and PODOPLANIN. Stained viable cells were sorted on a fluorescently activated cell sorter (FACS) to separate the CD34LowCD31PosVEGFR-3PosPODOPLANINPos LM LEC population from other endothelial and non-endothelial cells. The sorted LM LECs were cultured and expanded on fibronectin-coated flasks for further experimental use.

The goal of this protocol is to isolate lymphatic endothelial cells lining human lymphatic malformation cyst-like vessels and foreskins using fluorescence-activated cell sorting (FACS). Subsequent cell culturing and expansion of these cells permits a new level of experimental sophistication for genetic, proteomic, functional and cell differentiation studies.

人淋巴管内皮细胞中的多参​​数荧光激活细胞分选分离

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity ...

Isolation and Flow Cytometric Analysis of Human Endocervical Gamma Delta T Cells

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore essential for understanding pathogenicity of different pathogens including HIV. Gamma delta (GD) T cells are the prototype of &#39;unconventional&#39; T cells and represent a relatively small subset of T cells defined by their expression of heterodimeric T-cell receptors (TCRs) composed of gamma and delta chains. This sets them apart from the classical and much better known CD4+ helper T cells and CD8+ cytotoxic T cells that are defined by alpha-beta TCRs. GD T cells often show tissue-specific localization and are enriched in epithelium. GD T cells orchestrate immune responses in inflammation, tumor surveillance, infectious disease, and autoimmunity.
Here, we present a method to reproducibly isolate and analyze human endocervical intraepithelial GD T lymphocytes. We have used endocervical cytobrush samples from women participating in the Women&#39;s Interagency HIV Infection Study (WIHS). Knowledge about GD T cells interactions during conditions in which there is an insult to the vaginal mucosal could be applied to any clinical study in which mucosal vulnerability is addressed, including the development of vaginal microbicides.In addition, knowledge about mucosal GD T cell responses has potential for application of GD T cell-based immune therapy in treating infectious diseases.

We describe here an isolation method to obtain human endocervical intraepithelial lymphocytes for the analysis of intraepithelial gamma delta T cells. This protocol can be extended for the purification of endocervical gamma delta T cells by magnetic beads or by cell sorting.

人类宫颈的γδT细胞的分离和流式细胞仪分析

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore ...

免疫与感染

An Efficient Method for Extracting Human Fallopian Tube Epithelia for Single-cell Analyses

Human fallopian tubes are intrinsic to reproduction. The purpose of the fallopian tubes is to allow for the transit of sperm, the ovum, and if fertilization is successful, the embryo. The epithelial cells that line the inner surface of fallopian tubes are integral to both normal and abnormal fallopian tube processes, including disease initiation. After menopause, fallopian tubes cease to have a significant role in the body and the intraepithelial cell makeup changes. We describe a method in which these epithelial cells can be isolated from fresh fallopian tubes with minimal stromal cell contamination into a single-cell suspension. These cells can be grown in culture or used for further analysis such as flow cytometry and single-cell RNA sequencing. This isolation protocol can be achieved in 4-6 h and yields viable cells that can be used for immediate downstream analysis. This efficient protocol facilitates the isolation of fallopian tube epithelial cells with an enriched epithelial population.

This protocol describes a one-day method for the isolation of human fallopian tube epithelial cells. Isolated epithelial cells can be plated in 2-dimensional (2D) culture or dissociated into single-cell suspensions and utilized in downstream experiments, including flow cytometry and single-cell RNA sequencing.

提取人输卵管上皮细胞用于单细胞分析的有效方法

Human fallopian tubes are intrinsic to reproduction. The purpose of the fallopian tubes is to allow for the transit of sperm, the ovum, and if ...

Ex Vivo Infection of Human Lymphoid Tissue and Female Genital Mucosa with Human Immunodeficiency Virus 1 and Histoculture

Histocultures allow studying intercellular interactions within human tissues, and they can be employed to model host-pathogen interactions under controlled laboratory conditions. Ex vivo infection of human tissues with human immunodeficiency virus (HIV), among other viruses, has been successfully used to investigate early disease pathogenesis, as well as a platform to test the efficacy and toxicity of antiviral drugs. In the present protocol, we explain how to process and infect with HIV-1 tissue explants from human tonsils and cervical mucosae, and maintain them in culture on top of gelatin sponges at the liquid-air interface for about two weeks. This non-polarized culture setting maximizes access to nutrients in culture medium and oxygen, although progressive loss of tissue integrity and functional architectures remains its main limitation. This method allows monitoring HIV-1 replication and pathogenesis using several techniques, including immunoassays, qPCR, and flow cytometry. Of importance, the physiologic variability between tissue donors, as well as between explants from different areas of the same specimen, may significantly affect experimental results. To ensure result reproducibility, it is critical to use an adequate number of explants, technical replicates, and donor-matched control conditions to normalize the results of the experimental treatments when compiling data from multiple experiments (i.e., conducted using tissue from different donors) for statistical analysis.

Ex Vivo Infection of Human Lymphoid Tissue and Female Genital Mucosa with Human Immunodeficiency Virus 1 and Histoculture

Infection of human tissues with human immunodeficiency virus (HIV) ex vivo provides a valuable 3D model of virus pathogenesis. Here, we describe a protocol to process and infect tissue specimens from human tonsils and female genital mucosae with HIV-1 and maintain them in culture at the liquid-air interface.

体内人淋巴组织和女性生殖器粘膜与人体免疫机能丧失病毒1和 Histoculture 的感染

Histocultures allow studying intercellular interactions within human tissues, and they can be employed to model host-pathogen interactions under ...

Isolation of Dendritic Cells from the Human Female Reproductive Tract for Phenotypical and Functional Studies

The characterization of the human dendritic cells (DCs) resident in mucosal tissues is challenging due to the difficulty in obtaining samples, and the low numbers of DCs present per tissue. Yet, as the phenotype and function of DCs is modified by the tissue environment, it is necessary to analyze tissue resident DC populations, since blood derived DCs incompletely reflect the complexities of DCs in tissues. Here we present a protocol to isolate DCs from the human female reproductive tract (FRT) using hysterectomy specimens that allows both phenotypical and functional analyses. The protocol consists of tissue digestion to generate a single cell mixed cell suspension, followed by positive magnetic bead selection. Our tissue digestion protocol does not cleave surface markers, which allows phenotypical and functional analysis of DCs in the steady state, without overnight incubation or cell activation. This protocol can be adapted for the isolation of other immune cell types or isolation of DCs from other tissues.

We describe here a method to isolate and purify dendritic cells from different anatomical compartments in the human female reproductive tract for the evaluation of their phenotypical and functional characteristics. This method can be adapted to isolate other immune cells or dendritic cells from other mucosal tissues.

人雌性生殖道树突状细胞表型鉴定的分离及功能研究

The characterization of the human dendritic cells (DCs) resident in mucosal tissues is challenging due to the difficulty in obtaining samples, and the low ...

Isolation of Uterine Innate Lymphoid Cells for Analysis by Flow Cytometry

Described here is a simple method to isolate and phenotype mouse group 1 uterine innate lymphoid cells (g1 uILCs) from individual pregnant uterus by flow cytometry. The protocol describes how to set up time mating to obtain multiple synchronous dams, the mechanical and enzymatic digestion of the pregnant uterus, the staining of single-cell suspensions, and a FACS strategy to phenotype and discriminate g1 uILCs. Although this method inevitably loses the spatial information of cellular distribution within the tissue, the protocol has been successfully applied to determine uILC heterogeneity, their response to maternal and foetal factors affecting pregnancy, their gene expression profile, and their functions.

This is a method to isolate uterine lymphoid cells from both pregnant and non-pregnant mice. This method can be used for multiple downstream applications such as FACS phenotyping, cell sorting, functional assays, RNA-seq, and proteomics. The protocol here demonstrates how to phenotype group 1 uterine innate lymphoid cells by flow cytometry.

分离子宫先天淋巴细胞，用于流式细胞术分析

Described here is a simple method to isolate and phenotype mouse group 1 uterine innate lymphoid cells (g1 uILCs) from individual pregnant uterus by flow ...

Flow Cytometric Analysis of Biomarkers for Detecting Human Sperm Functional Defects

The conventional semen parameter analysis is widely used to assess male fertility. However, studies have found that ~15% of infertile patients show no abnormalities in conventional semen parameters. Additional technologies are needed to explain the idiopathic infertility and detect subtle sperm defects. Currently, biomarkers of sperm function, including sperm apoptosis, mitochondrial membrane potential (MMP), and DNA damage, reveal sperm physiology at the molecular level and are capable of predicting male fertility.
With flow cytometry (FCM) techniques, each of these markers can be rapidly, accurately, and precisely measured in human semen samples, but time costs substantially increase and results could be obstructed if all the biomarkers need to be tested with a single cytometer. In this protocol, after collection and immediate incubation at 37 &#176;C for liquefication, semen samples were further analyzed for sperm apoptosis using Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) staining. The MMP was labeled with 5,5&#8242;,6,6&#8242;-tetrachloro-1,1&#8242;,3,3&#8242;-tetraethyl-benzimidazolylcarbocyanine iodide (JC-1) probe, and DNA damage was assessed using the sperm chromatin structure assay (SCSA) with acridine orange (AO) staining. Thus, flow cytometric analysis of sperm function markers can be a practical and reliable toolkit for the diagnosis of infertility and evaluation of sperm function at both bench and bed.

The present protocol provides a practical solution that allows for the measurement of apoptosis, mitochondrial membrane potential, and DNA damage in human sperm with a single cytometer.

用于检测人类精子功能缺陷的生物标志物的流式细胞术分析

The conventional semen parameter analysis is widely used to assess male fertility. However, studies have found that ~15% of infertile patients show no ...

收集，分离，和人类宫颈样品的流式细胞仪分析

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用于检测人类精子功能缺陷的生物标志物的流式细胞术分析

收集，分离，和人类宫颈样品的流式细胞仪分析

摘要

探索更多视频

此视频中的章节

正常结肠和结直肠癌患者内皮细胞的分离--一种改进的协议

从临床样本中自动分离和收集癌症相关物质

两种方法从子宫切除标本小学建立人子宫内膜基质细胞

人淋巴管内皮细胞中的多参​​数荧光激活细胞分选分离

人类宫颈的γδT细胞的分离和流式细胞仪分析

提取人输卵管上皮细胞用于单细胞分析的有效方法

体内人淋巴组织和女性生殖器粘膜与人体免疫机能丧失病毒1和 Histoculture 的感染

人雌性生殖道树突状细胞表型鉴定的分离及功能研究

分离子宫先天淋巴细胞，用于流式细胞术分析

用于检测人类精子功能缺陷的生物标志物的流式细胞术分析

人淋巴管内皮细胞中的多参数荧光激活细胞分选分离