我们感谢她在取得临床标本的宝贵援助洛娜伦肖（爱丁堡乳腺癌单位）。 肿瘤物质的使用被批准的洛锡安研究伦理委员会（08/S1101/41）和实验癌症医学中心项目（爱丁堡）的主持下获得的。 这项工作是由苏格兰拨款委员会的支持。 ADL的是一个吴霭仪黄油Reekie信托临床研究员。

1。从鼠尾中提取胶原蛋白<ol><li>将在70％的乙醇新鲜冷冻老鼠尾巴。 </li><li> Deglove覆皮肤，用手术刀暴露筋。 </li><li>远离尾和70％的乙醇消毒地带筋。 </li><li>称量收集的肌腱和转移到乙酸（1G肌腱至250ml 0.5M醋酸）。混合48小时在4 ° C。 </li><li>离心机在10000克肌腱/醋酸酸混合为30min，弃去沉淀。 </li><li>添加一个同等体积的10％（W / V）氯化钠去上清液，并允许混合一夜之间站在4 ° C。 </li><li> 10,000收集的丰富的胶原蛋白，不溶于底层为进一步30分钟离心G. </li><li>重悬在0.25米乙酸胶原蛋白丰富的材料在4 ° C，并在4 ° C，连续3天对1:1000醋酸透析，不断变化的，每天两次的透析缓冲。 </li><li>进一步胶原溶液消毒离心（20000克为2小时）和存储在4 ° C。 </li><li>除了一个1mg/ml的股票浓度无菌1:1000醋酸稀释胶原。 </li></ol> 2。 3D含量创作 3D检测一个细胞系的实验基础上，此前公布的2。 <ol><li>多个核心活检收获同意在浸润性乳腺癌的治疗手术切除时患者。 </li><li>用眼，划分到1毫米3片段使用手术刀的核心。修剪和丢弃宏观独特的脂肪。 </li><li>沉浸在MEGM完整的媒体碎片。组织可以存储在这种状态下，以1小时。 </li><li>要创建的胶原蛋白凝胶，混合冰1mg/ml的鼠尾胶原，1:1000过滤醋酸，10倍的DMEM/F12和0.22M的NaOH的3:5:1:1的比例，以创建最后的胶原蛋白浓度为0.3毫克/毫升。 </li><li>进入孵化器的24孔板和地方的个别井注入胶原蛋白的混合物1.2毫升在37℃放置10min C至启动胶凝。 </li><li> 10min后，从孵化器中取出24孔板。胶原蛋白凝胶上的肿瘤块，轻轻推到中心使用一个加入10μl枪头凝胶。 24孔板返回为1小时的孵化器。 </li><li>对于ER +肿瘤，补充MEGM完整的媒体与β-雌二醇浓度3.2x10 -10米一旦凝胶检测凝胶为1小时，仔细地介绍每孔培养基1.2毫升。最后的β-雌二醇测定的浓度范围内，将equilibriate 1.6x10 -10米复述生理雌激素水平在乳房组织3中发现。 </li><li>运行每口井的内缘周围的加入10μl枪头，释放每个凝胶圆周，从而使每个凝胶媒体内自由浮动。返回的24孔板的孵化器。 </li><li>交易所补充媒体的两倍，直到实验终止每周。消除媒体和添加福尔马林固定液终止实验。 </li></ol> 3。光学投影层析成像抗体染色，在制剂的含量测定我们已经通过先前公布的方法，为整个样本染色 4 。 <ol><li>福尔马林固定标本用PBS 30分钟洗净。脱水凝胶逐步在PBS中含0.05％吐温20（0.05％PBST）甲醇稀释：33％，66％和100％≥15分钟，在每一步。 </li><li>孵育组织在新鲜配制的甲醇：二甲亚砜：30％H 2 O 2在2时01分03秒的比例，在室温为24小时（15％V / V H 2 O 2）淬火的自体荧光。 </li><li>甲醇30分钟，洗净样品两次。冻结的样品为-80 °彗星5次，每次至少1小时，回到室温（RT），以确保在组织的部位较深，抗原呈现的访问。 </li><li>组织补充水分0.05％PBST以甲醇作为稀释剂（33％，66％和100％为15分钟，在每一步）。 </li><li>座在24小时封锁的解决方案（10％在0.05％PBST血清）的组织。 </li><li>与第一抗体孵育组织确定上皮的内容，使用的1:500稀释浓度在阻断含有5％二甲基亚砜为48小时的解决方案1.5毫升的兔抗细胞角蛋白。 </li><li>洗四次0.05％PBST 30分钟每个样品。 </li><li>与荧光二抗（1:100羊抗兔Alexa555）1.5毫升阻塞的解决方案，其中包含48小时5％DMSO稀释，孵育组织。 </li><li>样品洗净，再与0.05％PBST 4次各为30分钟。 </li></ol> 4。光学投影层析成像光学投影层析成像是一个以前开发的显微技术，生产厚度高达15毫米的5荧光生物标本的高分辨率三维图像。 <ol><li>安装的Stained在1％低熔点琼脂糖凝胶为24小时和100％甲醇脱水。清除BABB溶液（1:2苯甲醇，苯甲酸苄酯）的标本为48小时和巴勒斯坦被占领土3001M扫描仪（Bioptonics）进行扫描。 </li><li>这两个目标和自体荧光捕获使用Cy3标记过滤设置（励磁545nm/30纳米七十五分之六百一十纳米），发射和GFP1过滤器设置（励磁425nm/40纳米，发射LP475纳米）。 </li></ol> 5。重建和三维定量<ol><li>由被占领土获得的角预测可重构使用NRecon软件（SkyScan）创建一个截面切片通过一系列的入侵检测。该软件可免费下载<a href="http://www.skyscan.be/products/downloads.htm">http://www.skyscan.be/products/downloads.htm</a> 。 </li><li>分析使用Volocity软件（珀金埃尔默）的重建数据。免费演示软件可供下载， <a href="http://www.cellularimaging.com/products/Volocity/demo/">在</a> http://www.cellularimaging.com/products/Volocity/demo/ 。 </li><li>为了量化上皮细胞角蛋白染色量，设置一个最小的体素强度阈值，以减少在目标通道的背景荧光干扰。我们定义区在9000这个门槛经验使用在测量中的应用可用参数的列表，我们量化与原有的核心（在autofluorescent通道划分）的上皮连续性量，这使我们能够量化不同上皮生长量肿瘤样本。 </li></ol> 6。代表性的成果： 在检测的发展，共52乳腺癌活检一个广泛的病理标本已被使用。其中&lt;10％（5 / 52）未能提供至少一个可行的入侵检测。检测失败，是因为无论是细菌性上部感染或不足肿瘤活检材料。因此检测中的应用不仅限于一个具体亚型乳腺癌。 固定后20天胶原文化的典型分析（图1），并随后为角蛋白染色。 Volocity软件能够识别上皮大部分与原有的核心和侵入细胞的分离群体持续和量化每卷分别。 <img src="/files/ftp_upload/3085/3085fig1.jpg" alt="图1" /> 图1。侵袭实验，固定和量化。检测支持从多个亚型乳腺癌样本：A）ER + B）的HER2 +和C）雌激素受体。

<ol>
	<li>Lopez-Garcia, M. A., Geyer, F. C., Lacroix-Triki, M., Marchio, C., Reis-Filho, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breast+cancer+precursors+revisited:+molecular+features+and+progression+pathways.+Histopathology.">Breast cancer precursors revisited: molecular features and progression pathways. Histopathology.</a> 57, 171-192 (2010).</li><li>Sabeh, F., Shimizu-Hirota, R., Weiss, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protease-dependent+versus+-independent+cancer+cell+invasion+programs:+three-dimensional+amoeboid+movement+revisited.">Protease-dependent versus -independent cancer cell invasion programs: three-dimensional amoeboid movement revisited.</a> J Cell Biol. 185, 11-19 (2009).</li><li>Dabrosin, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+extracellular+local+levels+of+estradiol+in+normal+breast+in+vivo+during+the+luteal+phase+of+the+menstrual+cycle.">Increased extracellular local levels of estradiol in normal breast in vivo during the luteal phase of the menstrual cycle.</a> J Endocrinol. 187, 103-108 (2005).</li><li>Alanentalo, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tomographic+molecular+imaging+and+3D+quantification+within+adult+mouse+organs.">Tomographic molecular imaging and 3D quantification within adult mouse organs.</a> Nat Methods. 4, 31-33 (2007).</li><li>Sharpe, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+projection+tomography+as+a+tool+for+3D+microscopy+and+gene+expression+studies.">Optical projection tomography as a tool for 3D microscopy and gene expression studies.</a> Science. 296, 541-545 (2002).</li><li>Hidalgo-Carcedo, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collective+cell+migration+requires+suppression+of+actomyosin+at+cell-cell+contacts+mediated+by+DDR1+and+the+cell+polarity+regulators+Par3+and+Par6.">Collective cell migration requires suppression of actomyosin at cell-cell contacts mediated by DDR1 and the cell polarity regulators Par3 and Par6.</a> Nat Cell Biol. 13, 49-58 (2011).</li><li>Katz, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+gene+on+the+HER2+amplicon,+C35,+is+an+oncogene+in+breast+cancer+whose+actions+are+prevented+by+inhibition+of+Syk.">A gene on the HER2 amplicon, C35, is an oncogene in breast cancer whose actions are prevented by inhibition of Syk.</a> Br J Cancer. 103, 401-410 (2010).</li><li>Timpson, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+Regulation+of+RhoA+Activity+during+Pancreatic+Cancer+Cell+Invasion+Driven+by+Mutant+p53.">Spatial Regulation of RhoA Activity during Pancreatic Cancer Cell Invasion Driven by Mutant p53.</a> Cancer Res. 71, 747-757 (2011).</li></ol>

SEW公司和JF是由MRC技术，这是商业化Bioptonics OPT的扫描仪的聘用。其他作者申报任何利益冲突。

胶原蛋白为基础的分析，研究肿瘤细胞株的行为是目前广泛使用的6,7,8。然而，这些实验都没有充分反映在肿瘤及其周围环境的复杂性。在这项研究中，我们显示，人类乳腺癌的材料，可用于以类似的方式， 体外 。此外，OPT是一个有用的工具来刻画乳腺癌的3D扩展。我们发现，这项技术的主要限制是肿瘤材料的可用性（单核心活检是足够4-6实验）。另一个限制因素是结构的入侵只是发生在约40％的检测。为了克服这个问题，我们会定期添加任何7天左右所需的治疗，所有检测明显增长。 在未来，可以延长使用OPT的，也发现大量变更 ，体外，药物治疗造成的。这反过来又可以促进更多的个性化的癌症治疗，独立的动物模型。目前，我们正在探索ER +肿瘤他莫昔芬体外的反应。

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录号 </td></tr><tr><td> MEGM完成</td><td>龙沙</td><td> CC - 3150 </td></tr><tr><td> DMEM/F12 </td><td> Sigma - Aldrich公司</td><td> D8900 10X1L </td></tr><tr><td> β-雌二醇</td><td> Sigma - Aldrich公司</td><td> E8875 - 250毫克</td></tr><tr><td>兔抗细胞角蛋白</td><td> Dako公司</td><td> Z0622 </td></tr><tr><td>羊抗兔Alexa555 </td><td> Invitrogen公司</td><td> A21422 </td></tr></tbody></table>所有其他的标准试剂均购自Sigma - Aldrich公司。

long-term culture of human breast cancer specimens and their analysis using optical projection tomography

乳腺癌是在西方世界造成的死亡率。它是公认的乳腺癌扩散，首先在本地和远端后，是在患者预后的主要因素。乳腺癌的实验系统依靠从原发肿瘤或胸腔积液通常派生的细胞株。两大障碍阻碍了这项研究：（一）一些已知的子类型的乳腺癌患者（尤其是预后不良的管腔乙肿瘤）没有代表在当前行的集合;（二）肿瘤微环境的影响通常不考虑。 我们展示一个小学文化的所有子类型的乳房癌标本的技术。这是通过使用三维（3D）文化系统中，小块的肿瘤是我垫在软大鼠胶原嵌入式。在2-3周之内，肿瘤细胞扩散到胶原蛋白，形成类似人类tumours1观察的各种结构。可行的脂肪细胞，上皮细胞和成纤维细胞内原有的核心，在组织学上可见一斑。 squamoid形态的恶性上皮细胞进行了论证，侵犯到周围的胶原质。这些细胞内，核异型明显，核分裂和凋亡小体。 我们所采用的光学投影层析成像（OPT），三维成像技术，以量化肿瘤的扩散程度文化。我们使用的OPT，测量肿瘤的文化体积，参数常规测量过程中的新辅助治疗的乳腺癌患者，以评估药物治疗的反应。 在这里，我们提供一个机会，文化的人类乳腺肿瘤，无分型的偏见和量化那些体内的扩散。这种方法可用于在未来量化在原发肿瘤的药物敏感性。这可以提供一个比目前使用的细胞株的预测模型。

Watch this Scientific Journal Video about Long-term Culture of Human Breast Cancer Specimens and Their Analysis Using Optical Projection Tomography at JoVE.com

Long-term Culture of Human Breast Cancer Specimens and Their Analysis Using Optical Projection Tomography

我们已经开发出一种基于胶原蛋白<em&gt;在体外</em&gt;促进所有乳腺癌亚型样本的增殖和侵袭的实验。利用光学投影层析成像，立体显微镜技术，可视化和量化肿瘤扩展。此法可用于量化个别肿瘤样本中的药物反应。

长期培养的人类乳腺癌标本，并利用光学投影层析成像分析

Breast cancer is a leading cause of mortality in the Western world. It is well established that the spread of breast cancer, first locally and later ...

long-term-culture-human-breast-cancer-specimens-their-analysis-using

Research

JoVE Journal

Biology

14.6K 次观看.  University of Edinburgh. 我们已经开发出一种基于胶原蛋白在体外促进所有乳腺癌亚型样本的增殖和侵袭的实验。利用光学投影层析成像，立体显微镜技术，可视化和量化肿瘤扩展。此法可用于量化个别肿瘤样本中的药物反应。

视频: Long-term Culture of Human Breast Cancer Specimens and Their Analysis Using Optical Projection Tomography

Long-term Imaging Mammalian Cells using Wide-Field Microscopy

长期使用宽视场显微镜的成像哺乳动物细胞

科研

生物学

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

一个神经元和星形胶质细胞共培养检测神经毒性的高含量分析

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

Born Normalization for Fluorescence Optical Projection Tomography for Whole Heart Imaging

Optical projection tomography is a three-dimensional imaging technique that has been recently introduced as an imaging tool primarily in developmental biology and gene expression studies. The technique renders biological sample optically transparent by first dehydrating them and then placing in a mixture of benzyl alcohol and benzyl benzoate in a 2:1 ratio (BABB or Murray s Clear solution). The technique renders biological samples optically transparent by first dehydrating them in graded ethanol solutions then placing them in a mixture of benzyl alcohol and benzyl benzoate in a 2:1 ratio (BABB or Murray s Clear solution) to clear. After the clearing process the scattering contribution in the sample can be greatly reduced and made almost negligible while the absorption contribution cannot be eliminated completely. When trying to reconstruct the fluorescence distribution within the sample under investigation, this contribution affects the reconstructions and leads, inevitably, to image artifacts and quantification errors.. While absorption could be reduced further with a permanence of weeks or months in the clearing media, this will lead to progressive loss of fluorescence and to an unrealistically long sample processing time. This is true when reconstructing both exogenous contrast agents (molecular contrast agents) as well as endogenous contrast (e.g. reconstructions of genetically expressed fluorescent proteins).

We suggest a Born normalized approach for Optical Projection Tomography (BnOPT) that accounts for the absorption properties of imaged samples to obtain accurate and quantitative fluorescence tomographic reconstructions. We use the proposed algorithm to reconstruct the fluorescence molecular probe distribution within small animal organs.

生于正常化荧光整个心脏成像的光学投影层析成像

Optical projection tomography is a three-dimensional imaging technique that has been recently introduced as an imaging tool primarily in developmental ...

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate daughter cells that retain the stem cell phenotype and transit-amplifying cells (TA cells) that migrate from the stem cell niche, undergo rapid proliferation and terminally differentiate to repopulate the tissue.
Epithelial stem cells have been identified in the epidermis, hair follicle, and intestine as cells with a high in vitro proliferative potential and as slow-cycling label-retaining cells in vivo 1-3. Adult, tissue-specific stem cells are responsible for the regeneration of the tissues in which they reside during normal physiologic turnover as well as during times of stress 4-5. Moreover, stem cells are generally considered to be multi-potent, possessing the capacity to give rise to multiple cell types within the tissue 6. For example, rodent hair follicle stem cells can generate epidermis, sebaceous glands, and hair follicles 7-9. We have shown that stem cells from the human hair follicle bulge region exhibit multi-potentiality 10.
Stem cells have become a valuable tool in biomedical research, due to their utility as an in vitro system for studying developmental biology, differentiation, tumorigenesis and for their possible therapeutic utility. It is likely that adult epithelial stem cells will be useful in the treatment of diseases such as ectodermal dysplasias, monilethrix, Netherton syndrome, Menkes disease, hereditary epidermolysis bullosa and alopecias 11-13. Additionally, other skin problems such as burn wounds, chronic wounds and ulcers will benefit from stem cell related therapies 14,15. Given the potential for reprogramming of adult cells into a pluripotent state (iPS cells)16,17, the readily accessible and expandable adult stem cells in human skin may provide a valuable source of cells for induction and downstream therapy for a wide range of disease including diabetes and Parkinson's disease.

A rapid, robust way of isolating viable adult epithelial stem cells from human skin is described. The method utilizes enzymatic digestion of skin collagen matrix , followed by plucking of hair follicles and isolation of single cell suspensions or tissue fragments for cell culture.

成人从人体皮肤的上皮干细胞的分离和培养

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate ...

Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

Epithelial ovarian cancer is a leading cause of female cancer mortality in the United States. In contrast to other women-specific cancers, like breast and uterine carcinomas, where death rates have fallen in recent years, ovarian cancer cure rates have remained relatively unchanged over the past two decades 1. This is largely due to the lack of appropriate screening tools for detection of early stage disease where surgery and chemotherapy are most effective 2, 3. As a result, most patients present with advanced stage disease and diffuse abdominal involvement. This is further complicated by the fact that ovarian cancer is a heterogeneous disease with multiple histologic subtypes 4, 5. Serous ovarian carcinoma (SOC) is the most common and aggressive subtype and the form most often associated with mutations in the BRCA genes. Current experimental models in this field involve the use of cancer cell lines and mouse models to better understand the initiating genetic events and pathogenesis of disease 6, 7. Recently, the fallopian tube has emerged as a novel site for the origin of SOC, with the fallopian tube (FT) secretory epithelial cell (FTSEC) as the proposed cell of origin 8, 9. There are currently no cell lines or culture systems available to study the FT epithelium or the FTSEC. Here we describe a novel ex vivo culture system where primary human FT epithelial cells are cultured in a manner that preserves their architecture, polarity, immunophenotype, and response to physiologic and genotoxic stressors. This ex vivo model provides a useful tool for the study of SOC, allowing a better understanding of how tumors can arise from this tissue, and the mechanisms involved in tumor initiation and progression.

Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

The fallopian tube (FT) is emerging as an alternative site of origin for serous ovarian carcinoma (SOC). This protocol describes a novel method for the isolation and ex vivo culture of fallopian tube epithelial cells. This system recapitulates the in vivo epithelium and allows the study of SOC pathogenesis.

前初级人类输卵管上皮细胞的体外培养

Epithelial ovarian cancer is a leading cause of female cancer mortality in the United States. In contrast to other women-specific cancers, like breast and ...

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish water and sediment. The choice of biological indicators must be based on reliable scientific knowledge and, possibly, on the availability of standardized procedures. In this article, we present a standardized protocol that used the marine crustacean Artemia to evaluate the toxicity of chemicals and/or of marine environmental matrices. Scientists propose that the brine shrimp (Artemia) is a suitable candidate for the development of a standard bioassay for worldwide utilization. A number of papers have been published on the toxic effects of various chemicals and toxicants on brine shrimp (Artemia). The major advantage of this crustacean for toxicity studies is the overall availability of the dry cysts; these can be immediately used in testing and difficult cultivation is not demanded1,2. Cyst-based toxicity assays are cheap, continuously available, simple and reliable and are thus an important answer to routine needs of toxicity screening, for industrial monitoring requirements or for regulatory purposes3. The proposed method involves the mortality as an endpoint. The numbers of survivors were counted and percentage of deaths were calculated. Larvae were considered dead if they did not exhibit any internal or external movement during several seconds of observation4. This procedure was standardized testing a reference substance (Sodium Dodecyl Sulfate); some results are reported in this work. This article accompanies a video that describes the performance of procedural toxicity testing, showing all the steps related to the protocol.

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

This study concerns the development and standardization of a valuable methodological protocol to determine long-term (14 days) lethal toxicity exerted by chemical substances, industrial wastewater or sewage and liquid environmental samples on the saltwater crustacean, Artemia franciscana.

致命的甲壳类动物长期毒性试验卤虫franciscana</em

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish ...

Serial Enrichment of Spermatogonial Stem and Progenitor Cells (SSCs) in Culture for Derivation of Long-term Adult Mouse SSC Lines

Spermatogonial stem and progenitor cells (SSCs) of the testis represent a classic example of adult mammalian stem cells and preserve fertility for nearly the lifetime of the animal. While the precise mechanisms that govern self-renewal and differentiation in vivo are challenging to study, various systems have been developed previously to propagate murine SSCs in vitro using a combination of specialized culture media and feeder cells1-3.
Most in vitro forays into the biology of SSCs have derived cell lines from neonates, possibly due to the difficulty in obtaining adult cell lines4. However, the testis continues to mature up until ~5 weeks of age in most mouse strains. In the early post-natal period, dramatic changes occur in the architecture of the testis and in the biology of both somatic and spermatogenic cells, including alterations in expression levels of numerous stem cell-related genes. Therefore, neonatally-derived SSC lines may not fully recapitulate the biology of adult SSCs that persist after the adult testis has reached a steady state.
Several factors have hindered the production of adult SSC lines historically. First, the proportion of functional stem cells may decrease during adulthood, either due to intrinsic or extrinsic factors5,6. Furthermore, as with other adult stem cells, it has been difficult to enrich SSCs sufficiently from total adult testicular cells without using a combination of immunoselection or other sorting strategies7. Commonly employed strategies include the use of cryptorchid mice as a source of donor cells due to a higher ratio of stem cells to other cell types8. Based on the hypothesis that removal of somatic cells from the initial culture disrupts interactions with the stem cell niche that are essential for SSC survival, we previously developed methods to derive adult lines that do not require immunoselection or cryptorchid donors but rather employ serial enrichment of SSCs in culture, referred to hereafter as SESC2,3.
The method described below entails a simple procedure for deriving adult SSC lines by dissociating adult donor seminiferous tubules, followed by plating of cells on feeders comprised of a testicular stromal cell line (JK1)3. Through serial passaging, strongly adherent, contaminating non-germ cells are depleted from the culture with concomitant enrichment of SSCs. Cultures produced in this manner contain a mixture of spermatogonia at different stages of differentiation, which contain SSCs, based on long-term self renewal capability. The crux of the SESC method is that it enables SSCs to make the difficult transition from self-renewal in vivo to long-term self-renewal in vitro in a radically different microenvironment, produces long-term SSC lines, free of contaminating somatic cells, and thereby enables subsequent experimental manipulation of SSCs.

Serial Enrichment of Spermatogonial Stem and Progenitor Cells SSCs in Culture for Derivation of Long-term Adult Mouse SSC Lines

A simple method to derive and maintain spermatogonial stem and progenitor cell lines from adult mice is presented here. The method utilizes feeder cells originating from the somatic cell compartment of the adult mouse testis. This technique is applicable to common mouse strains, including transgenic, knock-out, and knock-in mice.

在文化序列富集的精原干细胞和祖细胞（SSCS）推导长期成年小鼠SSC线

Spermatogonial stem and progenitor cells (SSCs) of the testis represent a classic example of adult mammalian stem cells and preserve fertility for nearly ...

Detecting Somatic Genetic Alterations in Tumor Specimens by Exon Capture and Massively Parallel Sequencing

Efforts to detect and investigate key oncogenic mutations have proven valuable to facilitate the appropriate treatment for cancer patients. The establishment of high-throughput, massively parallel "next-generation" sequencing has aided the discovery of many such mutations. To enhance the clinical and translational utility of this technology, platforms must be high-throughput, cost-effective, and compatible with formalin-fixed paraffin embedded (FFPE) tissue samples that may yield small amounts of degraded or damaged DNA. Here, we describe the preparation of barcoded and multiplexed DNA libraries followed by hybridization-based capture of targeted exons for the detection of cancer-associated mutations in fresh frozen and FFPE tumors by massively parallel sequencing. This method enables the identification of sequence mutations, copy number alterations, and select structural rearrangements involving all targeted genes. Targeted exon sequencing offers the benefits of high throughput, low cost, and deep sequence coverage, thus conferring high sensitivity for detecting low frequency mutations.

We describe the preparation of barcoded DNA libraries and subsequent hybridization-based exon capture for detection of key cancer-associated mutations in clinical tumor specimens by massively parallel "next generation" sequencing. Targeted exon sequencing offers the benefits of high throughput, low cost, and deep sequence coverage, thus yielding high sensitivity for detecting low frequency mutations.

由外显子捕获和大规模平行测序检测体细胞遗传学改变在肿瘤标本

Efforts  to detect and investigate key oncogenic mutations have proven valuable to  facilitate the appropriate treatment for cancer patients. The ...

Multilayer Mounting for Long-term Light Sheet Microscopy of Zebrafish

Light sheet microscopy is the ideal imaging technique to study zebrafish embryonic development. Due to minimal photo-toxicity and bleaching, it is particularly suited for long-term time-lapse imaging over many hours up to several days. However, an appropriate sample mounting strategy is needed that offers both confinement and normal development of the sample. Multilayer mounting, a new embedding technique using low-concentration agarose in optically clear tubes, now overcomes this limitation and unleashes the full potential of light sheet microscopy for real-time developmental biology.

The development of zebrafish can be followed over days with light sheet microscopy when embryos are embedded in optically clear polymer tubes with low-concentration agarose.

多层安装于斑马鱼的长期光表镜

Light sheet microscopy is the ideal imaging technique to study zebrafish embryonic development. Due to minimal photo-toxicity and bleaching, it is ...

Isolation, Transfection, and Long-Term Culture of Adult Mouse and Rat Cardiomyocytes

Ex vivo culture of the adult mammalian cardiomyocytes (CMs) presents the most relevant experimental system for the in vitro study of cardiac biology. Adult mammalian CMs are terminally differentiated cells with minimal proliferative capacity. The post-mitotic state of adult CMs not only restricts cardiomyocyte cell cycle progression but also limits the efficient culture of CMs. Moreover, the long-term culture of adult CMs is necessary for many studies, such as CM proliferation and analysis of gene expression.
The mouse and the rat are the two most preferred laboratory animals to be used for cardiomyocyte isolation. While the long-term culture of rat CMs is possible, adult mouse CMs are susceptible to death and cannot be cultured more than five days under normal conditions. Therefore, there is a critical need to optimize the cell isolation and long-term culture protocol for adult murine CMs. With this modified protocol, it is possible to successfully isolate and culture both adult mouse and rat CMs for more than 20 days. Moreover, the siRNA transfection efficiency of isolated CM is significantly increased compared to previous reports. For adult mouse CM isolation, the Langendorff perfusion method is utilized with an optimal enzyme solution and sufficient time for complete extracellular matrix dissociation. In order to obtain pure ventricular CMs, both atria were dissected and discarded before proceeding with the disassociation and plating. Cells were dispersed on a laminin coated plate, which allowed for efficient and rapid attachment. CMs were allowed to settle for 4-6 h before siRNA transfection. Culture media was refreshed every 24 h for 20 days, and subsequently, CMs were fixed and stained for cardiac-specific markers such as Troponin and markers of cell cycle such as KI67.

Here, we present a protocol for the isolation, transfection, and long-term culture of adult mouse and rat cardiomyocytes.

成年小鼠和大鼠心肌细胞的隔离、转染和长期培养

Ex vivo culture of the adult mammalian cardiomyocytes (CMs) presents the most relevant experimental system for the in vitro study of cardiac biology. ...