作者想感谢参与国际多中心验证研究修改表皮坐（5 ZEBET BfR的（德国），巴斯夫（德国），IIVS公司（美国），说＆劳基法（奥地利） ）。

一，组织空调 - 0天<ol><li>收到的表皮EPI - 200 -薛套件后，检查所有的完整套件组件（套件的详细信息，请参阅特定的试剂和仪器表）。 </li><li>每三个表皮组织，准备一个无菌6孔板与检测介质0.9毫升预填充。 </li><li>在无菌条件下，打开塑料袋中含有的24孔板中，含有表皮组织和取出的无菌纱布。 </li><li>用无菌镊子删除每个插入含有表皮组织和地方插入空的24孔板。在这一步，删除任何剩余的航运琼脂糖，坚持插入外侧轻轻印迹无菌滤纸上。 </li><li>接下来的5分钟之内，目视检查的组织。不要使用有缺陷的组织或组织与液体完全覆盖。 </li><li>使用无菌棉头棉签，精心组织表面干燥。尽量不要直接接触的组织 - 毛细管效应，足以消除组织表面的水分。 </li><li>组织表面干燥后，将三个组织的6孔0.9毫升检测介质预填充板的最上面一行。释放被困插入下方的空气气泡。 </li><li>将6孔板进入孵化器的插入包含为60 ± 5分钟预培养于37 ± 1 ° C，5 ± 1％的CO 2，95％RH（相对湿度）。 </li><li>在60 ± 5分钟前潜伏期月底，从上部井转移到6孔板较低井插入。放入孵化器板（37 ± 1 ° C，5 ± 1％的CO 2，95％相对湿度）过夜预培养（18 ± 3小时）。 </li><li>其余的检测介质放入冰箱（5 ± 3℃）。继隔夜预培养，可用于测试化学品的表皮组织。 注：预孵化过程可以缩短下旬组织交付的情况下（例如，如果组织周二周三抵达 ）。 </li></ol>二。化学品接触 - 第1天<ol><li>准备灌装只用0.9毫升，每口井的实验介质上排每一个测试化学品的6孔板。 </li><li>计划暴露于化学品前约5分钟，取出6孔板，从孵化器的预调节组织。 </li><li>评估组织的表面和消除任何水分，用无菌棉。 </li><li>测试材料代码或名称，标签所有6孔板盖。 </li><li>剂量在1分钟的时间间隔试品的组织，以方便曝光后试品的冲洗。保持剂量组织在无菌罩板，直到最后组织剂量。 </li><li>要测试液体化工品，免除直接顶上组织30μL的解决方案和地方组织表面上的尼龙网眼。如果有必要，轻轻网使用为首的玻璃巴斯德吸管灯泡的位置。不要按组织表面上。 </li><li>对于测试semisolids，使用正排量吸管免除30μL直接顶上组织。如果必要的传播与巴斯德吸管化学覆盖组织表面尽可能。 </li><li>对于固体材料，不久之前，应用程序的测试物质，滋润与25μL无菌DPBS组织表面。这将改善与测试化学品的组织表面的接触。填写细磨测试材料约25毫克25毫克校准尖锐的应用汤匙（或任何其他合适的工具）。应用组织表面的固体化学测试。如果有必要，可以使用一个灯泡为首的巴斯德吸管完全空勺子。轻轻摇动插入提高的固体表面上蔓延。 </li><li>糯一致性测试物质，努力形成直径约8毫米平坦的一块“如cookie”，在和地方组织之上，这是以前用无菌DPBS湿。为了提高测试的物质和组织之间的接触，权衡下来用不锈钢或塑料援助“曲奇”。 </li><li>应用作为阴性对照和5％作为阳性对照，以控制组织的SDS DPBS。 </li><li>服药后的最后组织，所有板块为35 ± 1分钟转移到培养箱（37 ± 1 ° C，5 ± 1％的CO 2，95％相对湿度）。 </li><li>后35 ± 1分钟的潜伏期在37 ° C删除所有的盘子从孵化器。放入无菌罩板和等待，直到60分钟的化学品接触的期限为第一剂量组织完成。 </li><li>之后开始洗涤过程使用一个插入每分钟的时间间隔（以insurE的为每个插入的总曝光时间是60分钟）。 </li><li>使用洗瓶冲洗用无菌DPBS的组织。 15次使用的DPBS络绎不绝填充插入组织和空，它（注：用尼龙网眼组织，洗净组织表面的5倍，并仔细指出，尖锐的钳取出网状之后，继续与其余10涤）。 </li><li> 15日冲洗后使用的洗瓶，完全淹没在150毫升DPBS插入的3倍和动摇删除测试材料的其余部分。 </li><li>最后，冲洗一次插入组织内，一旦从外面用无菌DPBS。 </li><li>取出轻轻晃动插入和印迹无菌吸墨纸任何多余的组织DPBS。 </li><li>抹杀的组织插入转移到新的6孔板以前检测介质预填充。必须为每个插入1分钟内完成所有这些步骤（14-18）。 </li><li>去年组织冲洗后，仔细地干各组织的表面，用无菌棉放倒棉签。 </li><li>在接下来的24 ± 2小时，在孵化器孵育组织（37 ± 1℃，5 ± 1％的CO 2，95％相对湿度） 。 注：每次检测技术人员不应该测试超过6人，包括在一个单一的测试运行（套）的阴性和阳性对照的测试物质，能够按照协议，如下所述</li></ol>三。换液 - 第2天<ol><li>后24 ± 2小时培养后，转移到下部的6孔板，用0.9毫升的媒体预填充的插入。额外的端点（细胞因子，趋化因子等）的分析，可以收集来自上游行的媒体。次要终点的分析是可选的。 </li><li>孵化后继续（37 ± 1℃，5 ± 1％的CO 2，95％RH）为18 ± 3小时。 </li></ol>四。 MTT可行性含量 - 3天<ol><li>标签两个24孔板的化学名称或代码。一个板块将成为组织孵化用MTT法和其他提取步骤。 </li><li>准备解冻的MTT液（5毫克/毫升）和MTT法稀释剂稀释MTT溶液。 MTT溶液的最终浓度为1毫克/毫升。 </li><li>吸取300μLMTT溶液到每孔24孔板。 </li><li>从孵化器中删除的6孔板，印迹在吸墨纸插入底部，并转移到24孔板中，用MTT预填充。 </li><li>在孵化器将24孔板（37 ± 1℃，5 ± 1％的CO 2，95％相对湿度）孵育3小时± 5分钟。严格遵守3小时± 5分钟的孵化时间，以避免不同的MTT法读数偏差。 </li><li>当MTT法孵化完成后，用吸泵，轻轻吸出所有的水井的MTT介质。 </li><li>笔芯与DPBS井和吸一次。重复这个漂洗两次以上，并确保最后的愿望后，组织干。 </li><li>清洗后的转移插入到一个新的24孔板。沉浸轻轻移液2 mL的萃取溶液（异丙醇）插入到每个插入。插入上部边缘以上的水平将上升，从而完全淹没的组织。 </li><li>密封的24孔板中（例如，使用密封袋封口膜或），抑制萃取蒸发。温柔摇晃一盘摇床（〜120 RPM）从组织提取物在室温下至少2小时的MTT。也可用于无晃动隔夜提取。 </li><li>提取期间完成后，皮尔斯与注射针头，或串珠巴斯德吸管灯泡和允许提取运行以及从中插入插入。 </li><li>丢弃刺破插入吸管的解决方案，以及向上和向下三次，直到它是均匀的。 </li><li>对于每个组织，转移到一个96孔平底微孔板的两个紫色的甲臜解决方案200μL等份。根据这个视频协议所附的电子表格中的固定板设计的重复传输。对于空白，使用异丙醇。 </li><li>阅读MTT法提取的光密度（OD值）在96孔板分光光度计，使用波长570纳米（540-580）无参考过滤器。 </li><li>在电子表格自动计算的结果（图1）输入的结果。 </li><li>测试的化学物质是标有“刺激性”（R38或综合住户统计调查第2类），MTT法测定的％的组织活力，如果是&lt;50％，相对于阴性对照。 注意：MTT法是有毒的，所以处理时一定要戴防护手套。保护MTT和其所以由轻，因为MTT法lutions对光敏感。 MTT溶液配制后几个小时内使用，因为它随着时间的推移降低。 </li></ol>五，检测质量控制<ul><li>表皮的EPI - 200 -薛套件的验收标准： 在体外 RHE模型应该提供一致，高质量的数据，随着时间的推移，不只是在验证过程中（8） 。建议的指引，以确保长期测定的重现性和性能，包括“充分表征的细胞或组织，每批取样... ...性能，并经常使用的控制和基准化学品提供保证检测性能的一致性 “（9） 。除下文所述的检测验收标准，每个生产批号的表皮是对一个额外的基准化学制造商（1％Triton X - 100的的）测试的质量控制。需要的曝光时间，以减少组织的生存能力，50％Triton X - 100的的决心（ET - 50）。对于表皮，ET - 50年均值从5.9小时到7.5小时不等。自1996年以来，很多很多CV为阴性对照组织（即基线可行性的变异）下平均每年7.5％。 ET50值每批产品必须属于2标准偏差，以历史平均水平，成立于1996年（即ET - 50 = 6.7小时;接受度较低限额= 4.8h;上接受限制= 8.7h）TRITON X - 100 ET50被释放的制造商使用。 </li><li> EPI - 200 -薛检测验收标准1：阴性对照： 绝对负对照组（NC）组织中的MTT测试（与无菌DPBS处理）的外径是在测试实验室中获得后，运输和储存的程序和特定的使用条件下的组织活力的一个指标。检测符合验收标准，如果平均外径 570数控组织是≥1.0和≤2.5 。 </li><li> EPI - 200 - 2薛检测验收标准：阳性对照作为阳性对照（PC）的5％的SDS（H 2 O）的解决方案是使用和测试与试品同时。并行的手段，要在每个检测测试，但不超过一台电脑是每个测试日的PC。阳性对照的可行性，应在95％置信区间的历史数据。检测符合验收标准，如果PC的组织平均可行性表示为阴性对照组织中的％20％。 </li><li> EPI - 200 -薛检测验收标准3：标准偏差（SD） 预计从3单一组织所确定的平均可行性皮肤刺激性和组织的变化，因此复制应该是可接受的低水平。检测符合验收标准，如果从个人组织存活率相同处理的3％计算的SD复制&lt;18％。 </li></ul>代表性的成果<div style="border:1px #000000 solid; width:100%; padding-bottom:5px;"><img src="/files/ftp_upload/1366/1366fig1.jpg" alt="图1" /> <img src="/files/ftp_upload/1366/1366fig1_2.jpg" alt="图2" /> <img src="/files/ftp_upload/1366/1366fig1_3.jpg" alt="图3" /> </div> 图1 6测试文章，NC和PC控制的结果-部分自动化电子表格计算结果。试品的减少低于50％的组织可行性引用到NC的被列为刺激。测试进行了优化，以提供足够远从分类切断（50％的可行性），从而提高检测的鲁棒性的结果。 <div style="background-image:url(../index_RES_1me8j21/c1vzp1s1_strip23px.png); background-position:0 100%; background-repeat:repeat-x; height:24px; width:100%;"><div style="padding-left:26px; padding-right:4px; padding-top:4px; width:100%;"> 材料 </div></div><img src="/files/ftp_upload/1366/materialstable.jpg" alt="物料清单" />

<ol>
	<li>Kand&#225;rov&#225;, H., Liebsch, M., Genschow, E., Gerner, I. n. g. r. i. d., Traue, D., Slawik, B., Spielmann, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+the+EpiDerm+Test+Protocol+for+the+upcoming+ECVAM+Validation+Study+on+In+Vitro+Skin+Irritation+Tests.">Optimization of the EpiDerm Test Protocol for the upcoming ECVAM Validation Study on In Vitro Skin Irritation Tests.</a> ALTEX. 21, 107-114 (2004).</li><li>Kand&#225;rov&#225;, H., Liebsch, M., Gerner, I., Schmidt, E., Genschow, E., Traue, D., Spielmann, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EpiDerm+Skin+Irritation+Test+Protocol+-+Assessment+of+the+performance+of+the+optimized+test.">EpiDerm Skin Irritation Test Protocol - Assessment of the performance of the optimized test.</a> ATLA. 33, 351-367 (2005).</li><li>Kand&#225;rov&#225;, H., Hayden, P., Klausner, M., Kubilus, J., Kearney, P., Sheasgreen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Vitro+Skin+Irritation+Test:+Improving+the+Sensitivity+of+the+EpiDerm+Skin+Irritation+Test+Protocol.Accepted+for+publication.">In Vitro Skin Irritation Test: Improving the Sensitivity of the EpiDerm Skin Irritation Test Protocol.Accepted for publication.</a> ATLA. , (2009).</li><li>Spielmann, H., Hoffmann, S., Liebsch, M., Botham, P., Fentem, J., Eskes, C., Roguet, R., Cotovi, J., Cole, T., Worth, A., Heylings, J., Jones, P., Robles, C., Kand&#225;rov&#225;, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ECVAM+International+Validation+Study+on+In+Vitro+Tests+for+Acute+Skin+Irritation:+Report+on+the+Validity+of+the+EPISKIN+and+EpiDerm+Assays+and+on+the+Skin+Integrity+Function+Test.">The ECVAM International Validation Study on In Vitro Tests for Acute Skin Irritation: Report on the Validity of the EPISKIN and EpiDerm Assays and on the Skin Integrity Function Test.</a> ATLA. 35, 559-601 (2007).</li><li>Liebsch, M., Gamer, A., Curren, R., Frank, J., Genschow, E., Tharmann, J., Remmele, M., Bauer, B., Raabe, H., Barnes, N., Hilberer, A., Wilt, N., Lornejad-Sch&#228;fer, M. R., Sch&#228;fer, C., Spiller, E., Hayden, P., Kand&#225;rov&#225;, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Follow-up+validation+of+the+modified+EpiDerm+Skin+Irritation+Test+(SIT):+results+of+a+multicentre+study+of+twenty+reference+test+substances.">Follow-up validation of the modified EpiDerm Skin Irritation Test (SIT): results of a multicentre study of twenty reference test substances.</a> , (2008).</li><li>United Nations. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Globally Harmonized System of Classification and Labeling of Chemicals (GHS). , (2007).</li><li>ESAC. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Statement on the Scientific Validity of In-Vitro Tests for Skin Irritation Testing. , (2008).</li><li>Gupta, K., Rispin, A., Stitzel, K., Coecke, S., Harbell, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ensuring+quality+of+in+vitro+alternative+test+methods:+Issues+and+answers.">Ensuring quality of in vitro alternative test methods: Issues and answers.</a> Regul Toxicol Pharmacol. 43, 219-224 (2005).</li><li>Rispin, A., Stitzel, K., Harbell, J., Klausner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ensuring+quality+of+in+vitro+alternative+test+methods:+Current+practice.">Ensuring quality of in vitro alternative test methods: Current practice.</a> Regul Toxicol Pharmacol. 45, 97-103 (2006).</li></ol>

The authors are employees of MatTek corporation which produce reagents and tools used in this article.

在这段视频中，我们表现出的表皮皮肤刺激性试验（表皮薛）的化学品，包括化妆品和药品的成分在体外皮肤刺激测试开发和验证。执行此方法时，重要的是，在无菌条件下工作，并严格遵循验证协议，因为从协议的偏差可能会导致不同的结果。检测了一些修改是可能的，然而，协议的变化应直接讨论与作者。 这种方法唯一的限制是一个与MTT端点的测试物质可能会干扰。一个彩色的测试物质或直接降低MTT法（从而模仿脱氢酶活性的细胞线粒体）可能会干扰与MTT终点。然而，这些试验物质是一个问题，只有在MTT试验（即42小时后测试物质的暴露）足够数量的测试物质时间上仍存在（或）组织。在这不太可能发生，（真）代谢MTT还原和贡献，一个彩色的测试材料或测试材料（假）详细介绍了在检测标准作业程序提供一个程序可以通过量化的直接MTT还原的情况下MatTek公司

an in vitro skin irritation test (sit) using the epiderm reconstructed human epidermal (rhe) model

表皮皮肤刺激性试验（表皮薛）<sup&gt;（1,2,3）</sup&gt;和验证<sup&gt;（4,5）</sup&gt;为<em&gt;在体外</em&gt;化学品的皮肤刺激测试，包括化妆品和​​药品的成分。表皮薛利用3D<em&gt;在体外</em&gt;重建人表皮（RHE）模型表皮。本议定书中所描述的程序，允许综合住户统计调查“类的刺激和非刺激之间的歧视<sup&gt;（6）</sup&gt;。测试是在4天的时间内过程中，预培养，60分钟的曝光，42小时后孵化和MTT可行性实验。组织收到过夜预培养（0天）后，组织局部暴露试品（1天），它可以是液体，半固体，固体或蜡。三个组织是用于每个测试化学品，以及为阳性对照（5％AQ。SDS溶液），与阴性对照（DPBS）。化学品接触时间为60分钟，35分钟的组织孵化器保持在37 ° C。测试物质，然后从组织表面广泛的洗涤程序。组织将被抹杀，并转移到新鲜培养基。经过24小时的潜伏期（2天），中期交换。介质可以是保存的细胞因子或其他感兴趣的终点进一步分析。换液后，一个额外的18小时内组织培养。在整个42H后孵化（3天）结束时，组织转移到黄色的MTT溶液中孵育3小时。由此产生的蓝紫色的甲臜盐，形成主要由线粒体代谢，提取2小时使用异丙醇。用分光光度计测定提取的甲臜的光学密度。被列为一个相对阴性对照组织处理的组织活力，如果低于50％减少刺激性的化学品。这个过程可以被用来作为完全替代的，在体内兔皮肤刺激性试验危险化学品的识别和标签符合欧盟法规<sup&gt;（7）</sup&gt;。

Watch this Scientific Journal Video about An In Vitro Skin Irritation Test SIT using the EpiDerm Reconstructed Human Epidermal RHE Model at JoVE.com

An In Vitro Skin Irritation Test SIT using the EpiDerm Reconstructed Human Epidermal RHE Model

在这段视频中，我们展示了开发和验证的表皮皮肤刺激性试验（表皮薛）<em&gt;在体外</em&gt;化学品的皮肤刺激测试，包括化妆品和​​药品的成分。

一个<em在体外皮肤刺激测试（SIT），使用的表皮重建人表皮（RHE）型号

The EpiDerm Skin Irritation test (EpiDerm SIT) was developed (1,2,3) and validated (4,5) for in vitro skin irritation testing of chemicals, including ...

an-vitro-skin-irritation-test-sit-using-epiderm-reconstructed-human

Research

JoVE Journal

Biology

An In Vitro Skin Irritation Test (SIT) using the EpiDerm Reconstructed Human Epidermal (RHE) Model

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视频: An In Vitro Skin Irritation Test SIT using the EpiDerm Reconstructed Human Epidermal RHE Model

Christopher Hughes:  An in vitro model for the Study of Angiogenesis (Interview)

Christopher C.W. Hughes describes the utility of his culture system for studying angiogenesis in vitro.  He explains the importance of fibroblasts that secrete a critical, yet unidentified, soluble factor that allow endothelial cells to form vessels in culture that branch, form proper lumens, and undergo anastamosis.

Christopher Hughes:  An in vitro model for the Study of Angiogenesis Interview

Christopher C.W. Hughes describes the utility of his culture system for studying angiogenesis in vitro. He explains the importance of fibroblasts that secrete a critical, yet unidentified, soluble factor that allow endothelial cells to form vessels in culture that branch, form proper lumens, and undergo anastamosis.

克里斯托弗休斯：在体外血管生成的研究模型（面试）

Christopher C.W. Hughes describes the utility of his culture system for studying angiogenesis in vitro.  He explains the importance of fibroblasts that ...

科研

生物学

Mouse Epidermal Neural Crest Stem Cell (EPI-NCSC) Cultures

EPI-NCSC are remnants of the embryonic neural crest in an adult location, the bulge of hair follicles. They are multipotent stem cells that have the physiological property to generate a wide array of differentiated cell types, including neurons, nerve supporting cells, smooth muscle cells, bone/cartilage cells and melanocytes. EPI-NCSC are easily accessible in the hairy skin and can be isolated as a highly pure population of stem cells. This video provides a detailed protocol for preparing mouse EPI-NCSC cultures from whisker follicles. The whisker pad of an adult mouse is removed, and whisker follicles dissected. The follicles are then cut longitudinally and subsequently transversely above and below the bulge region. The bulge is removed from the collagen capsule and placed in a culture plate. EPI-NCSC start to emigrate from the bulge explants 3 to 4 days later.

Mouse Epidermal Neural Crest Stem Cell EPI-NCSC Cultures

Here we show our method to isolate mouse epidermal neural crest stem cells (EPI-NCSC). Technique involves micro-dissecting whisker follicles, isolating the bulge and placeing it into tissue culture. EPI-NCSC start to emigrate from bulge explants onto the substratum within 3 - 4 days.

小鼠表皮神经嵴干细胞（EPI的非公务员合约雇员）文化

EPI-NCSC are remnants of the embryonic neural crest in an adult location, the bulge of hair follicles. They are multipotent stem cells that have the ...

An Allele-specific Gene Expression Assay to Test the Functional Basis of Genetic Associations

The number of significant genetic associations with common complex traits is constantly increasing. However, most of these associations have not been understood at molecular level. One of the mechanisms mediating the effect of DNA variants on phenotypes is gene expression, which has been shown to be particularly relevant for complex traits1.
This method tests in a cellular context the effect of specific DNA sequences on gene expression. The principle is to measure the relative abundance of transcripts arising from the two alleles of a gene, analysing cells which carry one copy of the DNA sequences associated with disease (the risk variants)2,3. Therefore, the cells used for this method should meet two fundamental genotypic requirements: they have to be heterozygous both for DNA risk variants and for DNA markers, typically coding polymorphisms, which can distinguish transcripts based on their chromosomal origin (Figure 1). DNA risk variants and DNA markers do not need to have the same allele frequency but the phase (haplotypic) relationship of the genetic markers needs to be understood. It is also important to choose cell types which express the gene of interest. This protocol refers specifically to the procedure adopted to extract nucleic acids from fibroblasts but the method is equally applicable to other cells types including primary cells.
DNA and RNA are extracted from the selected cell lines and cDNA is generated. DNA and cDNA are analysed with a primer extension assay, designed to target the coding DNA markers4. The primer extension assay is carried out using the MassARRAY (Sequenom)5 platform according to the manufacturer's specifications. Primer extension products are then analysed by matrix-assisted laser desorption/ionization time of-flight mass spectrometry (MALDI-TOF/MS). Because the selected markers are heterozygous they will generate two peaks on the MS profiles. The area of each peak is proportional to the transcript abundance and can be measured with a function of the MassARRAY Typer software to generate an allelic ratio (allele 1: allele 2) calculation. The allelic ratio obtained for cDNA is normalized using that measured from genomic DNA, where the allelic ratio is expected to be 1:1 to correct for technical artifacts. Markers with a normalised allelic ratio significantly different to 1 indicate that the amount of transcript generated from the two chromosomes in the same cell is different, suggesting that the DNA variants associated with the phenotype have an effect on gene expression. Experimental controls should be used to confirm the results.

Genetic associations often remain unexplained at a functional level. This method aims to assess the effect of phenotype-associated genetic markers on gene expression by analyzing cells heterozygous for transcribed SNPs. The technology allows accurate measurement by MALDI-TOF mass spectrometry to quantify allele-specific primer extension products.

等位基因特异性基因表达检测，测试的遗传协会的功能基础

The number of significant genetic associations with common complex traits is constantly increasing. However, most of these associations have not been ...

Imaging C. elegans Embryos using an Epifluorescent Microscope and Open Source Software

Cellular processes, such as chromosome assembly, segregation and cytokinesis,are inherently dynamic. Time-lapse imaging of living cells, using fluorescent-labeled reporter proteins or differential interference contrast (DIC) microscopy, allows for the examination of the temporal progression of these dynamic events which is otherwise inferred from analysis of fixed samples1,2. Moreover, the study of the developmental regulations of cellular processes necessitates conducting time-lapse experiments on an intact organism during development. The Caenorhabiditis elegans embryo is light-transparent and has a rapid, invariant developmental program with a known cell lineage3, thus providing an ideal experiment model for studying questions in cell biology4,5and development6-9. C. elegans is amendable to genetic manipulation by forward genetics (based on random mutagenesis10,11) and reverse genetics to target specific genes (based on RNAi-mediated interference and targeted mutagenesis12-15). In addition, transgenic animals can be readily created to express fluorescently tagged proteins or reporters16,17. These traits combine to make it easy to identify the genetic pathways regulating fundamental cellular and developmental processes in vivo18-21. In this protocol we present methods for live imaging of C. elegans embryos using DIC optics or GFP fluorescence on a compound epifluorescent microscope. We demonstrate the ease with which readily available microscopes, typically used for fixed sample imaging, can also be applied for time-lapse analysis using open-source software to automate the imaging process.

Imaging C. elegans Embryos using an Epifluorescent Microscope and Open Source Software

The C. elegans embryo is a powerful system for studying cell biology and development. We present a protocol for live imaging of C. elegans embryos utilizing DIC optics or fluorescence using readily available epifluorescent microscopes and open-source software.

成像 C。线虫胚胎使用啶显微镜和开放源码软件

Cellular processes, such as chromosome assembly, segregation and cytokinesis,are inherently dynamic. Time-lapse imaging of living cells, using ...

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 ...

Differentiation of Newborn Mouse Skin Derived Stem Cells into Germ-like Cells In vitro

Studying germ cell formation and differentiation has traditionally been very difficult due to low cell numbers and their location deep within developing embryos. The availability of a "closed" in vitro based system could prove invaluable for our understanding of gametogenesis. The formation of oocyte-like cells (OLCs) from somatic stem cells, isolated from newborn mouse skin, has been demonstrated and can be visualized in this video protocol. The resulting OLCs express various markers consistent with oocytes such as Oct4 , Vasa , Bmp15, and Scp3. However, they remain unable to undergo maturation or fertilization due to a failure to complete meiosis. This protocol will provide a system that is useful for studying the early stage formation and differentiation of germ cells into more mature gametes. During early differentiation the number of cells expressing Oct4 (potential germ-like cells) reaches ~5%, however currently the formation of OLCs remains relatively inefficient. The protocol is relatively straight forward though special care should be taken to ensure the starting cell population is healthy and at an early passage.

Differentiation of Newborn Mouse Skin Derived Stem Cells into Germ-like Cells In vitro

In recent years the formation of germ cells using in vitro culture methods has been demonstrated using several different types of somatic stem cells. This article will visually present the differentiation of newborn mouse derived stem cells to early oocyte-like cells.

新生小鼠皮肤干细胞分化成生殖细胞<em在体外</em

Studying  germ cell formation and differentiation has traditionally been very difficult  due to low cell numbers and their location deep within developing ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

An In Vitro Model for Studying Cellular Transformation by Kaposi Sarcoma Herpesvirus

Kaposi sarcoma (KS) is an unusual tumor composed of proliferating spindle cells that is initiated by infection of endothelial cells (EC) with KSHV, and develops most often in the setting of immunosuppression. Despite decades of research, optimal treatment of KS remains poorly defined and clinical outcomes are especially unfavorable in resource-limited settings. KS lesions are driven by pathological angiogenesis, chronic inflammation, and oncogenesis, and various in vitro cell culture models have been developed to study these processes. KS arises from KSHV-infected cells of endothelial origin, so EC-lineage cells provide the most appropriate in vitro surrogates of the spindle cell precursor. However, because EC have a limited in vitro lifespan, and as the oncogenic mechanisms employed by KSHV are less efficient than those of other tumorigenic viruses, it has been difficult to assess the processes of transformation in primary or telomerase-immortalized EC. Therefore, a novel EC-based culture model was developed that readily supports transformation following infection with KSHV. Ectopic expression of the E6 and E7 genes of human papillomavirus type 16 allows for extended culture of age- and passage-matched mock- and KSHV-infected EC and supports the development of a truly transformed (i.e., tumorigenic) phenotype in infected cell cultures. This tractable and highly reproducible model of KS has facilitated the discovery of several essential signaling pathways with high potential for translation into clinical settings.

An In Vitro Model for Studying Cellular Transformation by Kaposi Sarcoma Herpesvirus

Kaposi sarcoma (KS) is a tumor induced by infection with the oncogenic virus human herpesvirus-8/KS herpesvirus (HHV-8/KSHV). The endothelial cell culture model described here is uniquely suited for studying the mechanisms by which KSHV transforms host cells.

一种用于研究卡波西肉瘤细胞转化的体外模型

Kaposi sarcoma (KS) is an unusual tumor composed of proliferating spindle cells that is initiated by infection of endothelial cells (EC) with KSHV, and ...

Isolation of Mouse Epidermal Keratinocytes and Their In Vitro Clonogenic Culture

The protocol described here is a reliable method of harvesting primary keratinocytes from adult female mice (54 &#177; 2 days old) yielding approximately 30 x 106 viable cells per mouse. Primary adult mouse keratinocytes are harvested from the dorsal skin of female mice. Male mice (~6 weeks old) can be used for keratinocyte harvesting depending on the requirements of the experiment. Euthanized mice are shaved and sterilized with serial washes in povidone iodine and ethanol solutions (70% alcohol). After disinfecting the mice, the dorsal skin is removed and the subcutaneous fat and muscle are removed with a scalpel and discarded. The skins are cut into small pieces and treated with a mild, low temperature trypsinization to detach the lower dermis from the epidermis. The scraped epidermises are stirred at low speed, filtered to remove the hairs, counted, and re-suspended in culture medium. This method provides an excellent single cell suspension of highly culturable cells for many downstream applications.

The goal of this protocol is to isolate epidermal keratinocytes from the dorsal skin of adult mice for a variety of downstream applications such as molecular biology, biochemistry, fluorescence activated cell sorting, and primary in vitro uses (e.g., clonogenic keratinocytes).

小鼠表皮角质细胞的分离及其体外克隆培养物

The protocol described here is a reliable method of harvesting primary keratinocytes from adult female mice (54 &#177; 2 days old) yielding approximately ...

A Murine Model of a Burn Wound Reconstructed with an Allogeneic Skin Graft

Trivial superficial wounds heal without complications by primary intention. Deep wounds, such as full thickness burns, heal by secondary intention and require surgical debridement and skin grafting. Successful integration of the donor graft into a recipient wound bed depends on timely recruitment of immune cells, robust angiogenic response and new extracellular matrix formation. The development of novel therapeutic agents, which target some key processes involved in wound healing, are hindered by the lack of reliable preclinical models with optimized objective assessment of wound closure. Here, we describe an inexpensive and reproducible model of experimental full thickness burn wound reconstructed with an allogeneic skin graft. The wound is induced on the dorsum surface of anaesthetized inbred wild type mice from the BALB/C and SKH1-Hrhr backgrounds. The burn is produced using a brass template measuring 10 mm in diameter, which is preheated to 80 &#176;C and delivered at a constant pressure for 20 s. Burn eschar is excised 24 hours after the injury and replaced with a full thickness graft harvested from the tail of a genetically similar donor mouse. No specialized equipment is required for the procedure and surgical techniques are straightforward to follow. The method may be effortlessly implemented and reproduced in most research settings. Certain limitations are associated with the model. Due to technical difficulties, the harvest of thinner split thickness skin grafts is not possible. The surgical method we describe here allows for the reconstruction of burn wounds using full thickness skin grafts. It may be used to carry out preclinical therapeutic testing.

The aim of this study was to develop a murine model of burn wound healing. A thermal burn was induced on the dorsal skin of mice using a preheated brass template. Burned tissue was debrided and overlaid with a skin graft harvested from the tail of a genetically similar donor mouse.

用异体皮肤移植重建的烧伤伤口的穆林模型

Trivial superficial wounds heal without complications by primary intention. Deep wounds, such as full thickness burns, heal by secondary intention and ...

一个皮肤刺激测试（SIT），使用的表皮重建人表皮（RHE）型号