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

Nanyang Technological University

Research

JoVE Journal

Biology

73.2K 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

Human ES cells: Starting Culture from Frozen Cells

Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock.  First, a one to two day old ten cm plate of  approximately one (to two) million irradiated mouse embryonic fibroblast feeder cells is rinsed with HuES media to remove residual serum and cell debris, and then HuES media added and left to equilibrate in the cell culture incubator.  A frozen vial of cells from long term liquid nitrogen storage or a -80C freezer is sourced and quickly submerged in a 37C water bath for quick thawing.  Cells in freezing media are then removed from the vial and placed in a large volume of HuES media.  The large volume of HuES media facilitates removal of excess serum and DMSO, which can cause HuES human embryonic stem cells to differentiate.  Cells are gently spun out of suspension, and then re-suspended in a small volume of fresh HuES media that is then used to seed the MEF plate.  It is considered important to seed the MEF plate by gently adding the HuES cells in a drop wise fashion to evenly disperse them throughout the plate.  The newly established HuES culture plate is returned to the incubator for 48 hrs before media is replaced, then is fed every 24 hours thereafter.

Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock.

人类胚胎干细胞：启动文化冷冻细胞

Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock.  First, a one to two day old ten cm plate of  ...

科研

生物学

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are derived from the blastocyst of the early embryo and are pluripotent in differentiative ability.  Their vast differentiative potential has made them the focus of much research centered on deducing how to coax them to generate clinically useful cell types.  The successful derivation of hematopoietic stem cells (HSC) from mouse ESC has recently been accomplished and can be visualized in this video protocol.  HSC, arguably the most clinically exploited cell population, are used to treat a myriad of hematopoietic malignancies and disorders.  However, many patients that might benefit from HSC therapy lack access to suitable donors.  ESC could provide an alternative source of HSC for these patients.  The following protocol establishes a baseline from which ESC-HSC can be studied and inform efforts to isolate HSC from human ESC.  In this protocol, ESC are differentiated as embryoid bodies (EBs) for 6 days in commercially available serum pre-screened for optimal hematopoietic differentiation.  EBs are then dissociated and infected with retroviral HoxB4.  Infected EB-derived cells are plated on OP9 stroma, a bone marrow stromal cell line derived from the calvaria of  M-CSF-/- mice, and co-cultured in the presence of hematopoiesis promoting cytokines for ten days.  During this co-culture, the infected cells expand greatly, resulting in the generation a heterogeneous pool of 100s of millions of cells.  These cells can then be used to rescue and reconstitute lethally irradiated mice.

This protocol details the derivation of transplantable hematopoietic stem cells from mouse embryonic stem cells (ESC) and their subsequent injection into lethally irradiated recipient mice. Briefly, ESC are differentiated as embryoid bodies, which are then infected with retroviral HoxB4 and co-cultured with OP9 stromal cells and hematopoietic cytokines.

从小鼠胚胎干细胞的造血干细胞的推导

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are ...

Preparation of Neuronal Cultures from Midgastrula Stage Drosophila Embryos

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos and their dechorionation using bleach are demonstrated. Using a glass pipet attached to a mouth suction tube, we illustrate the removal of all cells from single embryos. The method for dispersing cells from each embyro into a small (5 l) drop of medium on an uncoated glass coverslip is demonstrated. A view through the microscope at 1 hour after plating illustrates the preferred cell density. Most of the cells that survive when grown in defined medium are neuroblasts that divide one or more times in culture before extending neuritic processes by 12-24 hours. A view through the microscope illustrates the level of neurite outgrowth and branching expected in a healthy culture at 2 days in vitro. The cultures are grown in a simple bicarbonate based defined medium, in a 5% CO2 incubator at 22-24&deg;C. Neuritic processes continue to elaborate over the first week in culture and when they make contact with neurites from neighboring cells they often form functional synaptic connections. Neurons in these cultures express voltage-gated sodium, calcium, and potassium channels and are electrically excitable. This culture system is useful for studying molecular genetic and environmental factors that regulate neuronal differentiation, excitability, and synapse formation/function.

This video demonstrates the preparation of primary neuronal cultures from midgastrula stage Drosophila embryos. Views of live cultures show cells 1 hour after plating and differentiated neurons after 2 days of growth in a bicarbonate-based defined medium. The neurons are electrically excitable and form synaptic connections.

从Midgastrula舞台果蝇胚胎神经文化的制备

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos ...

Preparation of Single-Cell Suspensions from Mouse Spleen with the gentleMACS Dissociator

Single-cell suspensions are a prerequisite for experiments in cell separation, cell analysis and cell culture.  To avoid tedious and often painful manual dissociations the gentleMACS Dissociator allows one to dissociate tissue very efficiently under controlled and reproducible conditions.  The gentleMACS Dissociator can optimally dissociate mouse spleen, combining timesaving and standardization with user-safety.

This video describes how to dissociate mouse spleens using the gentleMACS Dissociator, an automated bench-top device that can mechanically disrupt tissues using special tubes to produce viable cell suspensions. Following dissociation, spleens are filtered, centrifuged, and resuspended for further applications.

This video describes a simple, time-saving technique for automated tissue dissociation using the gentleMACS Dissociator to prepare single-cell suspensions of mouse splenocytes.

gentleMACS Dissociator从小鼠脾脏单细胞悬浮液的制备

Single-cell suspensions are a prerequisite for experiments in cell separation, cell analysis and cell culture.  To avoid tedious and often painful manual ...

Preparation of Drosophila S2 cells for Light Microscopy

The ideal experimental system would be cheap and easy to maintain, amenable to a variety of techniques, and would be supported by an extensive literature and genome sequence database. Cultured Drosophila S2 cells, the product of disassociated 20-24 hour old embryos1, possess all these properties. Consequently, S2 cells are extremely well-suited for the analysis of cellular processes, including the discovery of the genes encoding the molecular components of the process or mechanism of interest. The features of S2 cells that are most responsible for their utility are the ease with which they are maintained, their exquisite sensitivity to double-stranded (ds)RNA-mediated interference (RNAi), and their tractability to fluorescence microscopy as either live or fixed cells.
S2 cells can be grown in a variety of media, including a number of inexpensive, commercially-available, fully-defined, serum-free media2. In addition, they grow optimally and quickly at 21-24&deg;C and can be cultured in a variety of containers. Unlike mammalian cells, S2 cells do not require a regulated atmosphere, but instead do well with normal air and can even be maintained in sealed flasks.
Complementing the ease of RNAi in S2 cells is the ability to readily analyze experimentally-induced phenotypes by phase or fluorescence microscopy of fixed or live cells. S2 cells grow in culture as a single monolayer but do not display contact inhibition. Instead, cells tend to grow in colonies in dense cultures. At low density, S2 cultures grown on glass or tissue culture-treated plastic are round and loosely-attached. However, the cytology of S2 cells can be greatly improved by inducing them to flatten extensively by briefly culturing them on a surface coated with the lectin, concanavalin A (ConA)3. S2 cells can also be stably transfected with fluorescently-tagged markers to label structures or organelles of interest in live or fixed cells. Therefore, the usual scenario for the microscopic analysis of cells is this: first, S2 cells (which can possess transgenes to express tagged markers) are treated by RNAi to eliminate a target protein(s). RNAi treatment time can be adjusted to allow for differences in protein turn-over kinetics and to minimize cell trauma/death if the target protein is important for viability. Next, the treated cells are transferred to a dish containing a coverslip pre-coated with conA to induce cells to spread and tightly adhere to the glass. Finally, cells are imaged with the researcher's choice of microscopy modes. S2 cells are particularly good for studies requiring extended visualization of live cells since these cells stay healthy at room temperature and normal atmosphere.

Preparation of Drosophila S2 cells for Light Microscopy

Drosophila Schneider (S2) cells are an increasingly popular system for the discovery and functional analysis of genes. Our goal is to describe some of the microscopic techniques that make S2 cells such an increasingly important experimental system.

制备果蝇 S2细胞，光学显微镜

The ideal experimental system would be cheap and easy to maintain, amenable to a variety of techniques, and would be supported by an extensive literature ...

Preparation of Quality Inositol Pyrophosphates

Myo-inositol is present in nature either unmodified or in more complex phosphorylated derivates. Of the latest, the two most abundant in eukaryotic cells are inositol pentakisphosphate (IP5) and inositol hexakisphosphate (phytic acid or IP6). IP5 and IP6 are the precursors of inositol pyrophosphate molecules that contain one or more pyrophosphate bonds1. Phosphorylation of IP6 generates diphoshoinositolpentakisphosphate (IP7 or PP-IP5) and bisdiphoshoinositoltetrakisphosphate (IP8 or (PP)2-IP4). Inositol pyrophosphates have been isolated from all eukaryotic organisms so far studied. In addition, the two distinct classes of enzymes responsible for inositol pyrophosphate synthesis are highly conserved throughout evolution2-4.
The IP6 kinases (IP6Ks) posses an enormous catalytic flexibility, converting IP5 and IP6 to PP-IP4 and IP7 respectively and subsequently, by using these products as substrates, promote the generation of more complex molecules5,6. Recently, a second class of pyrophosphate generating enzymes was identified in the form of the yeast protein VIP1 (also referred as PP-IP5K), which is able to convert IP6 to IP7 and IP87,8.
Inositol pyrophosphates regulate many disparate cellular processes such as insulin secretion9, telomere length10,11, chemotaxis12, vesicular trafficking13, phosphate homeostasis14 and HIV-1 gag release15. Two mechanisms of actions have been proposed for this class of molecules. They can affect cellular function by allosterically interacting with specific proteins like AKT16. Alternatively, the pyrophosphate group can donate a phosphate to pre-phosphorylated proteins17. The enormous potential of this research field is hampered by the absence of a commercial source of inositol pyrophosphates, which is preventing many scientists from studying these molecules and this new post-translational modification. The methods currently available to isolate inositol pyrophosphates require sophisticated chromatographic apparatus18,19. These procedures use acidic conditions that might lead to inositol pyrophosphate degradation20 and thus to poor recovery. Furthermore, the cumbersome post-column desalting procedures restrict their use to specialized laboratories.
In this study we describe an undemanding method for the generation, isolation and purification of the products of the IP6-kinase and PP-IP5-kinases reactions. This method was possible by the ability of polyacrylamide gel electrophoresis (PAGE) to resolve highly phosphorylated inositol polyphosphates20. Following IP6K1 and PP-IP5K enzymatic reactions using IP6 as the substrate, PAGE was used to separate the generated inositol pyrophosphates that were subsequently eluted in water.

Inositol pyrophosphates play an important role in human pathologies such cancer, diabetes and obesity; however, the exact mechanism of action is a matter of dispute. The lack of commercially available inositol pyrophosphates renders detailed studies problematic. Here we describe a simple protocol to produce and isolate milligrams of inositol pyrophosphates.

编制质量肌醇Pyrophosphates

Myo-inositol is present in nature either unmodified or in more complex phosphorylated derivates. Of the latest, the two most abundant in eukaryotic cells ...

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 ...

Preparation of Primary Myogenic Precursor Cell/Myoblast Cultures from Basal Vertebrate Lineages

Due to the inherent difficulty and time involved with studying the myogenic program in vivo, primary culture systems derived from the resident adult stem cells of skeletal muscle, the myogenic precursor cells (MPCs), have proven indispensible to our understanding of mammalian skeletal muscle development and growth. Particularly among the basal taxa of Vertebrata, however, data are limited describing the molecular mechanisms controlling the self-renewal, proliferation, and differentiation of MPCs. Of particular interest are potential mechanisms that underlie the ability of basal vertebrates to undergo considerable postlarval skeletal myofiber hyperplasia (i.e.&#160;teleost fish) and full regeneration following appendage loss (i.e.&#160;urodele amphibians). Additionally, the use of cultured myoblasts could aid in the understanding of regeneration and the recapitulation of the myogenic program and the differences between them. To this end, we describe in detail a robust and efficient protocol (and variations therein) for isolating and maintaining MPCs and their progeny, myoblasts and immature myotubes, in cell culture as a platform for understanding the evolution of the myogenic program, beginning with the more basal vertebrates. Capitalizing on the model organism status of the zebrafish (Danio rerio), we report on the application of this protocol to small fishes of the cyprinid clade Danioninae. In tandem, this protocol can be utilized to realize a broader comparative approach by isolating MPCs from the Mexican axolotl (Ambystomamexicanum) and even laboratory rodents. This protocol is now widely used in studying myogenesis in several fish species, including rainbow trout, salmon, and sea bream1-4.

In vitro culture systems have proven indispensible to our understanding of vertebrate myogenesis. However, much remains to be learned about nonmammalian skeletal muscle development and growth, particularly in basal taxa. An efficient and robust protocol for isolating the adult stem cells of this tissue, the myogenic precursor cells (MPCs), and maintaining their self-renewal, proliferation, and differentiation in a primary culture setting allows for the identification of conserved and divergent regulatory mechanisms throughout the vertebrate lineages.

原发性生肌前体细胞/成肌细胞从基底哺乳动物支系文化准备

Due to the inherent difficulty and time involved with studying the myogenic program in vivo, primary culture systems derived from the resident adult stem ...

Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs include modeling pathogenesis of human genetic diseases, autologous cell therapy after gene correction, and personalized drug screening by providing a source of patient-specific and symptom relevant cells. However, there are several hurdles to overcome, such as eliminating the remaining reprogramming factor transgene expression after human iPSCs production. More importantly, residual transgene expression in undifferentiated human iPSCs could hamper proper differentiations and misguide the interpretation of disease-relevant in vitro phenotypes. With this reason, integration-free and/or transgene-free human iPSCs have been developed using several methods, such as adenovirus, the piggyBac system, minicircle vector, episomal vectors, direct protein delivery and synthesized mRNA. However, efficiency of reprogramming using integration-free methods is quite low in most cases.
Here, we present a method to isolate human iPSCs by using Sendai-virus (RNA virus) based reprogramming system. This reprogramming method shows consistent results and high efficiency in cost-effective manner.

Here, we present our established method to reprogram human somatic cells into transgene-free human iPSCs with Sendai virus, which shows consistent outcome and enhanced efficiency.

从人类体细胞与仙台病毒高效的新一代人类诱导多能干细胞

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs ...

Cryo-electron Microscopy Specimen Preparation By Means Of a Focused Ion Beam

Here we present a protocol used to prepare cryo-TEM samples of Aspergillus niger spores, but which can easily be adapted for any number of microorganisms or solutions. We make use of a custom built cryo-transfer station and a modified cryo-SEM preparation chamber2. The spores are taken from a culture, plunge-frozen in a liquid nitrogen slush and observed in the cryo-SEM to select a region of interest. A thin lamella is then extracted using the FIB, attached to a TEM grid and subsequently thinned to electron transparency. The grid is transferred to a cryo-TEM holder and into a TEM for high resolution studies. Thanks to the introduction of a cooled nanomanipulator tip and a cryo-transfer station, this protocol is a straightforward adaptation to cryogenic temperature of the routinely used FIB preparation of TEM samples. As such it has the advantages of requiring a small amount of modifications to existing instruments, setups and procedures; it is easy to implement; it has a broad range of applications, in principle the same as for cryo-TEM sample preparation. One limitation is that it requires skillful handling of the specimens at critical steps to avoid or minimize contaminations.

Cryo Electron Microscopes, either Scanning (SEM) or Transmission (TEM), are widely used for characterization of biological samples or other materials with a high water content1. A SEM/Focused Ion Beam (FIB) is used to identify features of interest in samples and extract a thin, electron-transparent lamella for transfer to a cryo-TEM.

低温电子显微镜样品制备借助于聚焦离子束

Here we present a protocol used to prepare cryo-TEM samples of Aspergillus niger spores, but which can easily be adapted for any number of microorganisms ...