作者感谢克里斯Chartrand，HT（ASCP）展示了部分与AV标记的细胞块切割。

简介： 这是一个视频描述Shidham的细胞液基细胞学（LBC）使用HistoGelTM（Thermo Scientific的）（HG）的标本块制备方法。相对于其他随机方法，以下是本议定书的两个关键特性，准备从相对松散的细胞（1-5），单分散的hypocellular标本细胞块的。 <ol><li>该协议涉及的步骤集中沿平面平行的细胞块切割面的细胞。 </li><li>它还包括一个AV标记（图1b），灯塔般的黑暗，以符合以下目的两个： <ol style="list-style-type:lower-alpha;"><li>要可视化的水平，这些细胞都集中在。现在感兴趣的细胞与细胞块面积可以可视化的histotechnologist时，深色的灯塔是在切割过程中暴露。这种监控能力，防止切割的水平，大部分细胞或不切割样品细胞浓度最高水平太肤浅。 </li><li>作为一个不同的幻灯片上串行细胞块段的定位参考点。这个参考点作为一盏明灯，帮助查找特定的细胞或细胞与上海化学工业区的方法（6,7）的坐标免疫模式的评价。 </li></ol></li></ol>协议（图2） 样品制备。 <ol><li>转移剩余LBC宫颈细胞学标本平底玻璃试管（直径15mm x 45毫米）（图2.1至2.4）。玻璃管放入一个更大的塑料载体管（28 × 85毫米）和离心机。删除从承运人管玻璃底管，倒出上清液。 </li><li>玻璃管，然后封顶（在下一步的加热水，以防止溢出），并放在一个较大的平底托盘塑料管</li><li>托盘塑料管中的玻璃管，然后加盖，放置在离心机旋转杯，并没有固定的角杯，使细胞平底玻璃管垂直下降，并在1805纺G（3000 RPM的，转子半径17厘米）为5分钟（图2.5）。 </li><li>然后取出管垂直离心机和删除，而不会干扰细胞的沉淀颗粒小玻璃管载体较大的塑料管钳。 </li><li>与样品的玻璃管是不封顶的，浇灭了上清小心不要打扰底部泥沙细胞平层（图2.6）。 </li></ol>列入参考坐标AV标记此外凝胶<ol><li>一个黑暗的灯塔AV标记 （约2毫米× 2毫米大小，平面浮出水面，深色，sectionable材料的片段）（图3），增加一条，作为一个玻璃管的路标（图2.7 ）。 </li><li>熔点为10秒，在中等功率微波液化等分的汞。 </li><li>加入0.5毫升的熔融汞管，迅速与泥沙混合，并重述（图2.8）（不允许HG的开始巩固的情况下迅速着手进行下一步）。 </li><li>新增约2.5毫升的温水（45 ° C）水向承运人的塑料管（图2.9）。 </li><li>较小的上限玻璃管放在里面的塑料管，用温水。 （这一步是必要的，以防止在接下来的步骤巩固了HG）（图2.9）。 </li><li>承运人塑料管被放置在离心机（ 与旋转杯，并没有固定的角杯，使细胞平底玻璃管垂直下降），和G（3000 RPM的，转子半径17厘米纺在1805年五）分钟。这种离心步骤的目的是推动影音标记，并集中到一个层细胞接近最终石蜡包埋细胞块（图1d）的切割面。 </li><li>然后取出，轻轻地，垂直管的照顾，不要去打扰样品池底部的沉淀一层薄薄的离心机。 </li><li>较大的塑料管是不封顶的小玻璃管中删除一个镊子垂直，而不会干扰标本细胞的沉积层。 </li><li>小玻璃管中冷藏15分钟，冷却和巩固HG（图2.11）的垂直位置。 </li></ol>作为细胞的凝胶标本按钮块进行最终处理的去除<ol><li>凝固HG磁盘，在底层的集中/沉积物样本，被赶出平底玻璃管喷出通过23号针头与注射器（图2.12）10％的福尔马林。 </li><li>针插入凝固标本（图2.12）中汞光盘的边缘沿管端。 </li><li>该needle是旋转沿管的一侧，而甲醛是缓慢通过注射器推。伴随着深色灯塔AV标记集中在从平底的玻璃管（图2.12）标本中汞按钮分离结果。 </li><li>细胞块（标本细胞的凝胶按钮），然后放置在一个标签的磁带和组织处理提交给准备石蜡包埋细胞块（图2.13 ）。 </li></ol>嵌入和切割试样<ol><li>磁盘是嵌入在切割面黑暗中的灯塔标记侧（图1）石蜡。 </li><li>直到深色AV标记块切片，一盏明灯，是揭露和清晰可见。 </li><li>从这个层面上，它应该包含最分散的单细胞标本3到4微米的部分削减。 </li><li>部分收集玻片上作进一步的染色，免疫组织化学染色，或作为其它测试。对这些类型的幻灯片，用于安装部分的测试协议可能会有所不同。一般免疫，涂幻灯片，以防止在免疫步骤浮动和幻灯片的部分路段损失。 </li></ol> （按字母顺序）的缩写 ：CISH，显色原位杂交试验，鱼，荧光原位杂交试验FFPE，福尔马林固定石蜡包埋;活检，细针穿刺; HG，HistoGel™（Thermo Scientific的） ; LBC，液基细胞学检查; PCR聚合酶链反应; <img src="/files/ftp_upload/1316/1382fig1.jpg" alt="图1" /> 图1。细胞块的结构编制Shidham的协议。 <img src="/files/ftp_upload/1316/1382fig2.jpg" alt="图2" /> 图2。Shidham的协议，准备从LBC标本的细胞块的不同步骤的摘要。 <img src="/files/ftp_upload/1316/1382fig3.jpg" alt="图3" /> 图3。香蕉皮AV标记的制备。 <img src="/files/ftp_upload/1316/1382fig4.jpg" alt="图4" /> 图4。AV标记和无细胞块和部分比较。 <img src="/files/ftp_upload/1316/1382fig5.jpg" alt="图5" /> 图5比较有和没有AV标记的细胞块切片的细胞结构。

<ol>
	<li>Shidham, V. B., Epple, J., Shidham, V. B., Atkinson, B. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+14,+Appendix+I:+Collection+and+processing+of+effusion+fluids+for+cytopathologic+evaluation.">Chapter 14, Appendix I: Collection and processing of effusion fluids for cytopathologic evaluation.</a> Cytopathologic Diagnosis of Serous Fluids. , .</li><li>Varsegi, G., D'Amore, K., Shidham, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=p16INK4a+Immunocytochemistry+as+an+Adjunct+to+Cervical+Cytology+-+Potential+Reflex+Testing+on+Specially+Prepared+Cellblocks+from+Residual+Liquid+Based+Cytology+(LBC)+Specimens.">p16INK4a Immunocytochemistry as an Adjunct to Cervical Cytology - Potential Reflex Testing on Specially Prepared Cellblocks from Residual Liquid Based Cytology (LBC) Specimens.</a> Modern Pathology 22: 98th Annual Meeting of United States and Canadian Academy of Pathology. , 97a-97a (2009).</li><li>Nigro, K., Tynski, Z., Wasman, J., Abdul-Karim, F., Wang, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+cell+block+preparation+methods+for+nongynecologic+ThinPrep+specimens.">Comparison of cell block preparation methods for nongynecologic ThinPrep specimens.</a> Diagn Cytopathol. 35, 640-643 (2007).</li><li>Saleh, H. A., Hammoud, J., Zakaria, R., Khan, A. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+Thin-Prep+and+cell+block+preparation+for+the+evaluation+of+Thyroid+epithelial+lesions+on+fine+needle+aspiration+biopsy.">Comparison of Thin-Prep and cell block preparation for the evaluation of Thyroid epithelial lesions on fine needle aspiration biopsy.</a> CytoJournal. 5, 3-3 (2008).</li><li>Kyroudi, A., Paefthimiou, M., Symiakaki, H., Mentzelopoulou, P., Voulgaris, Z., Karakitsos, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increasing+diagnostic+accuracy+with+a+cell+block+preparation+from+thin-layer+endometrial+cytology:+a+feasibility+study.">Increasing diagnostic accuracy with a cell block preparation from thin-layer endometrial cytology: a feasibility study.</a> Acta Cytol. 50, 63-69 (2006).</li><li>Atkinson, B. F., Shidham, V. B., Atkinson, B. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+5:+Immunocytochemistry+of+effusion+fluids:+introduction+to+SCIP+approach+(Chapter+5.">Chapter 5: Immunocytochemistry of effusion fluids: introduction to SCIP approach (Chapter 5.</a> Cytopathologic Diagnosis of Serous Fluids. , (2007).</li><li>Shidham, V. B., Shidham, V. B., Atkinson, B. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+15,+Appendix+II:+Immunocytochemistry+of+effusions+processing+and+commonly+used+immunomarkers.">Chapter 15, Appendix II: Immunocytochemistry of effusions processing and commonly used immunomarkers.</a> Cytopathologic Diagnosis of Serous Fluids. , (2007).</li></ol>

细胞块是一个有价值的工具，评估各种细胞学标本（1）。最重要的是，除了建筑细节的标本，细胞块允许配套的研究，如免疫细胞化学，荧光/色原位杂交试验（FISH / CISH）和原位PCR的评价。多种方法制备细胞块。然而，这些最适合非妇科细胞学标本，其中含有较多的细胞和组织microfragments如活检抽吸和一些浆液性液体，如积液（1,3,4,5）。 ，由于细胞块，主要提供免疫评价的机会，他们的处理最好的福尔马林固定石蜡包埋（FFPE）组织的类似。最终，免疫细胞块切片评价后得到的结果进行了比较FFPE组织切片主要出版的文献。在协议的任何改变可能的妥协和否定了通过不同的固定剂和试剂序列比常规FFPE使用的其他处理的细胞块上所取得的成果的有效性。一些商业技巧，可能已通过其他固定液试剂曝光的曝光的协议处理的缺点。 细胞块准备的各种方法都提供相当数量的泥沙和组织碎片标本。主要支持一些凝胶或凝血原则，集中沉积物。机动按钮，然后像手术病理标本/活检的处理后，石蜡包埋。 所用的凝胶包括明胶，琼脂，纤维蛋白原/血浆凝血酶，和其他商业凝胶，如HG。浓度的方法不尽相同，从简单的泥沙离心造粒沿膜的不同类型细胞的浓度。例子包括：Milipore，collodin（Celloidin）袋或刮从细胞学涂片的玻片上（1）细胞。我们还评估了各种凝胶，通过尝试不同的琼脂和明胶组合。组合没有实现的坚定足够的一致性，以获得在一块标本嵌入细胞凝固凝胶容易机动光盘。 HG表现出适当的一致性和HG细胞块切片的免疫组化结果在我们的经验已经很好的。 液基细胞学（LBC）宫颈细胞学标本一般比非妇科标本较少的细胞，如上所述。此外，妇科LBC标本主要包含个别分散脱落宫颈粘膜表层细胞。由于这个原因，适当的细胞结构内的细胞块切片可能不能没有一种特殊的办法来实现。由于这些单分散的的块细胞成分可以不被的剖切期间histotechnologist看到，在该细胞开始出现在路段的水平不能赞赏并可能会丢失，或者由过去的水平切割与大多数细胞或不切割到足够深的层次最高浓度的样品细胞（图4）。 Shidham的协议地址使用嵌入中型和常规实验室设备（2）（图2）HG的这些问题都。 该协议涉及以下两个主要特点（图1）： <ol><li>相邻和平行的切割表面的细胞块（图5）在一个狭窄的飞机的单分散细胞的浓度和对齐。 </li><li>包括像一盏明灯，黑暗的 AV作为一个路标标记。这是实现以下功能的关键： <ol style="list-style-type:lower-alpha;"><li>确定和监测细胞集中在细胞块的水平。深色路标曝光突出分散的单细胞在细胞块的大部分预计将位于（图4）的水平，这可以防止过度切削 （切割过去的水平大部分细胞）或伤人 （不削减到足够深的层次最高浓度的样品细胞）的细胞块，并选择部分允许在相应的最高浓度的细胞块水平细胞。 </li><li>黑暗的部分彩色路标，也可作为一个参考点，进行调查和确定不同的序列在不同的幻灯片上的细胞块（图4）部分完全相同的细胞。同时解释和评估的坐标属性，特定的细胞由上海化学工业区的做法等immunoprofile遵循相同的细胞在不同的部分（6,7），这是非常重要的。 </li></ol></li></ol>虽然OUř协议标准化和液基宫颈细胞学标本报告，它也可以用于其他许多非妇科标本，如积液液，活检，刷检，囊肿内容等，以提高诊断率，此外AV标记将有助于改善上海化学工业区的方法应用在这些标本的细胞块切片的免疫组织化学评价。包埋剂，可能会被替换其他试剂与适当的修改，在有关步骤。 HG可能会被替换血浆（纤维蛋白原）在室温下（1）凝血酶凝胶。

cell block preparation from cytology specimen with predominance of individually scattered cells

这个视频演示Shidham的宫颈液基细胞学标本含有单独分散的细胞和小细胞群体的细胞块的制备方法。这种技术使用HistoGel（Thermo Scientific的）与传统的实验室设备。 使用细胞块切片是一种宝贵的的评价非妇科细胞学检查的辅助工具。他们使cytopathologist研究额外的形态标本的细节，包括病变的架构。最重要的是，他们允许为辅助研究的评价，如免疫细胞化学，原位杂交试验（FISH / CISH）和原位聚合酶链反应（PCR）。传统的细胞块制备技术大多被应用于非妇科细胞学标本，通常用于津液积液和细针穿刺活检。 液基宫颈标本相对较少，比他们的非妇科同行与许多单个细胞散细胞。正因为如此，足够的内细胞块切片的细胞​​结构是难以实现的。此外，histotechnologist切片块不能想像细胞在浓度最高的是在哪一级。因此，它是难以监测的适当水平，在这部分可以选择要进行测试，以玻片。因此，感兴趣的细胞与细胞块面积可能会被错过，无论是过去或切割不切割深度不够。目前“议定书”Shidham的方法解决这些问题。虽然这个协议是标准化和妇科液基细胞学标本报道，它也可以被应用于非妇科标本，如液体积液，活检，刷检，囊肿内容质量的提高细胞块切片的诊断材料等。

Watch this Scientific Journal Video about Cell Block Preparation from Cytology Specimen with Predominance of Individually Scattered Cells at JoVE.com

Cell Block Preparation from Cytology Specimen with Predominance of Individually Scattered Cells

Shidham准备从单独分散的细胞和小细胞群体的细胞学标本AV标记的细胞块的方法。

个别细胞散为主细胞学标本的细胞块的制备

This video demonstrates Shidham's method for preparation of cell blocks from liquid based cervicovaginal cytology specimens containing individually ...

cell-block-preparation-from-cytology-specimen-with-predominance

Research

JoVE Journal

Biology

73.4K 次观看.  University of Wisconsin - Milwaukee. Shidham准备从单独分散的细胞和小细胞群体的细胞学标本AV标记的细胞块的方法。

视频: Cell Block Preparation from Cytology Specimen with Predominance of Individually Scattered Cells

Preparation of Primary Acute Lymphoblastic Leukemia Cells in Different Cell Cycle Phases by Centrifugal Elutriation

The ability to synchronize cells has been central to advancing our understanding of cell cycle regulation. Common techniques employed include serum deprivation; chemicals which arrest cells at different cell cycle phases; or the use of mitotic shake-off which exploits their reduced adherence. However, all of these have disadvantages. For example, serum starvation works well for normal cells but less well for tumor cells with compromised cell cycle checkpoints due to oncogene activation or tumor suppressor loss. Similarly, chemically-treated cell populations can harbor drug-induced damage and show stress-related alterations. A technique which circumvents these problems is counterflow centrifugal elutriation (CCE), where cells are subjected to two opposing forces, centrifugal force and fluid velocity, which results in the separation of cells on the basis of size and density. Since cells advancing through the cycle typically enlarge, CCE can be used to separate cells into different cell cycle phases. Here we apply this technique to primary acute lymphoblastic leukemia cells. Under optimal conditions, an essentially pure population of cells in G1 phase and a highly enriched population of cells in G2/M phases can be obtained in excellent yield. These cell populations are ideally suited for studying cell cycle-dependent mechanisms of action of anticancer drugs and for other applications. We also show how modifications to the standard procedure can result in suboptimal performance and discuss the limitations of the technique. The detailed methodology presented should facilitate application and exploration of the technique to other types of cells.

This protocol describes the use of centrifugal elutriation to separate primary acute lymphoblastic leukemia cells into different cell cycle phases.

离心淘洗制备不同细胞周期的原发性急性淋巴细胞白血病细胞

The ability to synchronize cells has been central to advancing our understanding of cell cycle regulation. Common techniques employed include serum ...

科研

癌症研究

Micromanipulation of Circulating Tumor Cells for Downstream Molecular Analysis and Metastatic Potential Assessment

Blood-borne metastasis accounts for&#160;most cancer-related deaths and involves circulating tumor cells (CTCs) that are successful in establishing new tumors at distant sites. CTCs are found in the bloodstream of patients as single cells (single CTCs) or as multicellular aggregates (CTC clusters and CTC-white blood cell clusters), with the latter displaying a higher metastatic ability. Beyond enumeration, phenotypic and molecular analysis is extraordinarily important to dissect CTC biology and to identify actionable vulnerabilities. Here, we provide a detailed description of a workflow that includes CTC immunostaining and micromanipulation, ex vivo culture to assess&#160;proliferative and survival capabilities of individual cells, and in vivo metastasis-formation assays. Additionally, we provide a protocol to achieve the dissociation of CTC clusters into individual cells and the investigation of intra-cluster heterogeneity. With these approaches, for instance, we precisely quantify survival and proliferative potential of single CTCs and individual cells within CTC clusters, leading us to the observation that cells within clusters display better survival and proliferation in ex vivo cultures compared to single CTCs. Overall, our workflow offers a platform to dissect the characteristics of CTCs at the single cell level, aiming towards the identification of metastasis-relevant pathways and a better understanding of CTC biology.

Here, we present an integrated workflow to identify phenotypic and molecular features that characterize circulating tumor cells (CTCs). We combine live immunostaining and robotic micromanipulation of single and clustered CTCs with single cell-based techniques for downstream analysis and assessment of metastasis-seeding ability.

循环肿瘤细胞的微操作, 用于下游分子分析和转移电位评估

Blood-borne metastasis accounts for&#160;most cancer-related deaths and involves circulating tumor cells (CTCs) that are successful in establishing new ...

Cell Fractionation of U937 Cells by Isopycnic Density Gradient Purification

This protocol describes a method to obtain subcellular protein fractions from mammalian cells using a combination of detergents, mechanical lysis, and isopycnic density gradient centrifugation. The major advantage of this procedure is that it does not rely on the sole use of solubilizing detergents to obtain subcellular fractions. This makes it possible to separate the plasma membrane from other membrane-bound organelles of the cell. This procedure will facilitate the determination of protein localization in cells with a reproducible, scalable, and selective method. This method has been successfully used to isolate cytosolic, nuclear, mitochondrial, and plasma membrane proteins from the human monocyte cell line, U937. Although optimized for this cell line, this procedure may serve as a suitable starting point for the subcellular fractionation of other cell lines. Potential pitfalls of the procedure and how to avoid them are discussed as are alterations that may need to be considered for other cell lines.

This fractionation protocol will allow researchers to isolate cytoplasmic, nuclear, mitochondrial, and membrane proteins from mammalian cells. The latter two subcellular fractions are further purified via isopycnic density gradient.

通过等密度梯度纯化对U937细胞进行细胞分级分离

This protocol describes a method to obtain subcellular protein fractions from mammalian cells using a combination of detergents, mechanical lysis, and ...

生物化学

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.

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 ...

医学

Isolation and Characterization of a Head and Neck Squamous Cell Carcinoma Subpopulation Having Stem Cell Characteristics

Despite advances in the understanding of head and neck squamous cell carcinomas (HNSCC) progression, the five-year survival rate remains low due to local recurrence and distant metastasis. One hypothesis to explain this recurrence is the presence of cancer stem-like cells (CSCs) that present inherent chemo- and radio-resistance. In order to develop new therapeutic strategies, it is necessary to have experimental models that validate the effectiveness of targeted treatments and therefore to have reliable methods for the identification and isolation of CSCs. To this end, we present a protocol for the isolation of CSCs from human HNSCC cell lines that relies on the combination of two successive cell sortings performed by fluorescence activated cell sorting (FACS). The first one is based on the property of CSCs to overexpress ATP-Binding Cassette (ABC) transporter proteins and thus exclude, among others, vital DNA dyes such as Hoechst 33342. The cells sorted with this method are identified as a &#34;side population&#34; (SP). As the SP cells represent a low percentage (&#60;5%) of parental cells, a growing phase is necessary in order to increase their number before the second cell sorting. The next step allows for the selection of cells that possess two other HNSCC stem cell characteristics i.e. high expression level of the cell surface marker CD44 (CD44high) and the over-expression of aldehyde dehydrogenase (ALDHhigh). Since the use of a single marker has numerous limitations and pitfalls for the isolation of CSCs, the combination of SP, CD44 and ALDH markers will provide a useful tool to isolate CSCs for further analytical and functional assays requiring viable cells. The stem-like characteristics of CSCs was finally validated in vitro by the formation of tumorispheres and the expression of &#946;-catenin.

Understanding the role of cancer stem-like cells in tumor recurrence and resistance to therapies has become a topic of great interest in the last decade. This article describes the isolation and characterization of the sub-population of cancer stem-like cells from head and neck squamous carcinoma cell lines (HNSCC).

分离和表征头颈部鳞状细胞癌亚群具有干细胞特性

Despite advances in the understanding of head and neck squamous cell carcinomas (HNSCC) progression, the five-year survival rate remains low due to local ...

Chromosome Preparation From Cultured Cells

Chromosome (cytogenetic) analysis is widely used for the detection of chromosome instability. When followed by G-banding and molecular techniques such as fluorescence in situ hybridization (FISH), this assay has the powerful ability to analyze individual cells for aberrations that involve gains or losses of portions of the genome&#160;and rearrangements involving one or more chromosomes. In humans, chromosome abnormalities occur in approximately 1 per 160 live births1,2, 60-80% of all miscarriages3,4, 10% of stillbirths2,5, 13% of individuals with congenital heart disease6, 3-6% of infertility cases2, and in many patients with developmental delay&#160;and birth defects7. Cytogenetic analysis of malignancy is routinely used by researchers and clinicians, as observations of clonal chromosomal abnormalities have been shown to have both diagnostic and prognostic significance8,9.&#160; Chromosome isolation is invaluable for gene therapy and stem cell research of organisms including nonhuman primates and rodents10-13.
Chromosomes can be isolated from cells of live tissues, including blood lymphocytes, skin fibroblasts, amniocytes, placenta, bone marrow, and tumor specimens. Chromosomes are analyzed at the metaphase stage of mitosis, when they are most condensed and therefore more clearly visible. The first step of the chromosome isolation technique involves the disruption of the spindle fibers by incubation with Colcemid, to prevent the cells from proceeding to the subsequent anaphase stage. The cells are then treated with a hypotonic solution and preserved in their swollen state with Carnoy&#39;s fixative. The cells are then dropped on to slides and can then be utilized for a variety of procedures. G-banding involves trypsin treatment followed by staining with Giemsa to create characteristic light and dark bands. The same procedure to isolate chromosomes can be used for the preparation of cells for procedures such as fluorescence in situ hybridization (FISH), comparative genomic hybridization (CGH), and spectral karyotyping (SKY)14,15.

Chromosomes can be isolated from live cells such as lymphocytes or skin fibroblasts, and from organisms including humans or mice. These chromosome preparations can be further utilized for routine G-banding and molecular cytogenetic procedures such as fluorescence in situ hybridization (FISH), comparative genomic hybridization (CGH), and spectral karyotyping (SKY).

染色体制备培养细胞

Chromosome (cytogenetic) analysis is widely used for the detection of chromosome instability. When followed by G-banding and molecular techniques such as ...

生物学

An Alternative Approach for Sample Preparation with Low Cell Number for TEM Analysis

Transmission electron microscopy (TEM) provides details of the cellular organization and ultrastructure. However, TEM analysis of rare cell populations, especially cells in suspension such as hematopoietic stem cells (HSCs), remains limited due to the requirement of a high cell number during sample preparation. There are a few cytospin or monolayer approaches for TEM analysis from scarce samples, but these approaches fail to get significant quantitative data from the limited number of cells. Here, an alternative and novel approach for sample preparation in TEM studies is described for rare cell populations that enables quantitative analysis.
A relatively low cell number, i.e., 10,000 HSCs, was successfully used for TEM analysis compared to the millions of cells typically used for TEM studies. In particular, Evans blue staining was performed after paraformaldehyde-glutaraldehyde (PFA-GA) fixation to visualize the tiny cell pellet, which facilitated embedding in agarose. Clusters of numerous cells were observed in ultra-thin sections. The cells had a well preserved morphology, and the ultra-structural details of the Golgi complex and several mitochondria were visible. This efficient, easy and reproducible protocol allows sample preparation from a low cell number and can be used for qualitative and quantitative TEM analysis on rare cell populations from limited biological samples.

Here, we present a protocol to prepare samples with low cell numbers for transmission electron microscopy (TEM) analysis.

对于样品制备与TEM分析低细胞数量的另一种方法

Transmission electron microscopy (TEM) provides details of the cellular organization and ultrastructure. However, TEM analysis of rare cell populations, ...

用于 Multiome 10X 基因组学分析的高质量样品制备

High-Quality Brain and Bone Marrow Nuclei Preparation for Single Nuclei Multiome Assays

Single-cell analysis has become the approach of choice for unraveling the complexity of biological processes that require assessing the variability of individual cellular responses to treatment or infection with single-cell resolution. 
Many techniques for single-cell molecular profiling have been developed over the past 10 years, and several dedicated technologies have been commercialized. The 10X Genomics droplet-based single-cell profiling is a widespread technology that offers ready-to-use reagents for transcriptomic and multi-omic single-cell profiling. The technology includes workflows for single-cell and single-nuclei RNA sequencing (scRNA-Seq and snRNA-Seq, respectively), scATAC-Seq, single-cell immune profiling (BCR/TCR sequencing), and multiome. The latter combines transcriptional (scRNA-Seq) and epigenetic information (scATAC-Seq) coming from the same cell. 
The quality (viability, integrity, purity) of single-cell or single-nuclei suspensions isolated from tissues and analyzed by any of these approaches is critical for generating high-quality data. Therefore, the sample preparation protocols should be adapted to the particularities of each biological tissue and ensure the generation of high-quality cell and nuclei suspensions.
This article describes two protocols for preparing brain and bone marrow samples for the downstream multiome 10X Genomics pipeline. The protocols are performed stepwise and cover tissue dissociation, cell sorting, nuclei isolation, and quality control of prepared nuclei suspension that is used as starting material for cell partitioning and barcoding, library preparation, and sequencing. These standardized protocols produce high-quality nuclei libraries and robust and reliable data.

作者聚焦：通过单细胞分析推进生物医学研究 

The success of single-cell/single-nuclei transcriptomics and multi-omics largely depends on the quality of cells/nuclei. Therefore, isolating cells/nuclei from tissue and their purification must be highly standardized. This protocol describes the preparation of nuclei from the brain and bone marrow for downstream single-nuclei multiome assay.

用于单核多组学检测的高质量脑和骨髓核制备

Single-cell analysis has become the approach of choice for unraveling the complexity of biological processes that require assessing the variability of ...

个别细胞散为主细胞学标本的细胞块的制备

摘要

探索更多视频

离心淘洗制备不同细胞周期的原发性急性淋巴细胞白血病细胞

循环肿瘤细胞的微操作, 用于下游分子分析和转移电位评估

通过等密度梯度纯化对U937细胞进行细胞分级分离

收集，分离，和人类宫颈样品的流式细胞仪分析

分离和表征头颈部鳞状细胞癌亚群具有干细胞特性

染色体制备培养细胞

对于样品制备与TEM分析低细胞数量的另一种方法

用于单核多组学检测的高质量脑和骨髓核制备

用于单核多组学检测的高质量脑和骨髓核制备

用于单核多组学检测的高质量脑和骨髓核制备