J. Heijmans is supported by a stipendium from the Dutch cancer foundation (KFW). G.R. van den Brink is supported by funding from the European Research Council under the European Community&#8217;s Seventh Framework Program (FP7/2007-2013)/European Research Council grant agreement number 241344 and by a VIDI grant from the Netherlands Organization for Scientific Research (GvdB).

1.制备聚乙烯亚胺（PEI）作为转染试剂的<ol><li>溶解约150毫克的PEI入100毫升的H 2 O. </li><li>通过添加盐酸，直到解决方案变得清晰，搅拌至完全溶解调节溶液的pH值7.4。这可能需要10至60分钟，并添加水至1毫克/毫升的终浓度。 </li><li>当清晰，筛选在5毫升分装到无菌的0.22微米的过滤器和存储的解决方案，PEI在-80℃的冰柜。 </li></ol> 2.生产慢病毒颗粒的第1天: <ol><li>拆分HEK293T细胞，以60％-80％汇合162 平方厘米烧瓶或细胞系培养基大陪替氏培养皿（DMEM补充有10％FCS，1％青霉素/链霉素和2mM谷氨酰胺补充）。 </li></ol>第2天: <ol start="2"><li>制备含有45微克总质粒DNA加在一起的DNA转染溶液慢病毒载体的包装（7微克pVSVg的; 5微克PRSV转的13微克PMDL）的编码感兴趣感兴趣或shRNA基因的慢病毒载体和20微克。调整到使用的DMEM 1ml的体积。 </li><li>制备PEI的转染溶液加入90微升1mg / ml的PEI与930微升DMEM中，并孵育5分钟，在室温下进行。 </li><li>加入DNA转染的解决方案PEI。涡流或反转的次数，并孵育5分钟，在室温下，获得的DNA转染溶液。 </li><li>滴将2ml DNA转染溶液到HEK293T细胞孵育4小时，在湿润的细胞培养箱中于37℃。 </li><li> 4小时后，刷新培养基去除PEI。这是没有必要添加新介质之前洗涤细胞。 注:PEI是细胞毒性和孵育时间长于4小时可能造成伤害HEK293T细胞。 </li></ol>第4天: <ol start="7"><li>更换苏佩rnatant新的培养基。保持上清（含病毒）;这将在步骤2.10中使用。 </li><li>把上清液在15毫升烧瓶中。要去除死细胞，离心5分钟500×克。 </li><li>采用大60毫升注射器通过0.45微米的过滤器推上清。隔夜存放于4℃。 </li></ol>第5天: <ol start="10"><li>收集第二批上清液;离心和过滤器与步骤2.8-2.9。 </li><li>在超速离心90分钟，从池步2.9和超速离心管2.10和离心机上清50,000 XG。 </li><li>取出含有超速离心管非常仔细胶囊并放入层流罩，记住在离心机内的管的方向。 </li><li>打开胶囊保持超速离心管和倾析培养基小心以这样的方式，该粒料是在管的上侧。由于病毒丸粒可能难以显现，记住什么管侧的粒料将已经形成。取微量和除去介质的最后一个比特，同时小心不要搅动不透明棕色沉淀是在超速离心管的底部的侧可见。 </li><li>重悬该沉淀在500μl类器官培养基补充了10mM烟酰胺，10μMCHIR99021，10μMY27632和8微克/毫升聚凝胺。再悬浮在该介质是重要的，因为高滴度病毒用于直接转导组织体。 </li><li>可选:冻结病毒在这个媒介分装在两批250微升-80℃。 </li></ol>组织体3慢病毒转导第0天: <ol><li>拆分全0.95厘米2以及类器官两天转成新井之前的，旨在获得约50个小类器官。根据此前公布的方案1（ 图3A，B）分裂组织体。 </li><li>补充类器官用10μM的CHIR99021和10mM烟酰胺培养基，得到囊性增生过度隐窝（ 图3C）。 注:囊性隐窝将开发最好当新鲜分裂组织体生长在CHIR99021的存在。 </li></ol>第2天: <ol start="3"><li>收获组织体通过上下抽吸基质胶和介质，从而打乱了P1000微量的混合物。放置在一个15毫升管的混合物。 </li><li>使用巴斯德吸移管，其中所述远端开口已经下降了熔化进一步扰乱。离心机组织体以沉淀5分钟，在100×g下。 注:依赖于离心机的大小，​​使用不同的离心机的速度。有必要在其中找到破坏组织体从混合物中分离出的确切的速度。 </li><li>除去上清液，并加入500μl预热1X胰蛋白酶（类似于0.25％胰蛋白酶）的。悬浮类器官的胰蛋白酶和孵化3分钟在37℃水浴。 </li><li>通过加入3.5毫升细胞系培养基的灭活胰蛋白酶（DMEM补充有10％FCS，1％青霉素/链霉素和谷氨酰胺）。离心5分钟，在500×g。 </li><li>除去上清液留下沉淀在大约20微升培养基。传送在此最后少量介质的组织体成井在48孔板。 </li><li>加入高滴度慢病毒的转导中的步骤2.14，重悬描述和继续加强3.10。当遇到低转导效率，加强3.9可以添加。 注:为了有效地传导，一般加250微升的高滴度慢病毒1等分，以组织体的一个孔。这代表在本协议的第2步描述从单一生产的收获慢50％。 </li><li>任选:以提高转导效率，通过将48孔平板含有组织体在预温热离心机上32执行spinoculation6; C和旋转，在600×g离心1小时。 </li><li>把组织体病毒混合物在培养箱中孵育1小时，在37℃的培养箱中，以允许转导。 </li><li>任选:进一步提高转导效率，在一个培养箱中孵育组织体另外3小时，在37℃。 </li><li>加入1毫升组织体培养基和悬浮的组织体病毒混合物，在850×g离心转移到微量离心管中并离心以微量为5分钟以沉淀组织体。 </li><li>去除上清，悬浮颗粒在20微升冰冷的基质胶的。因为当它成为温暖的材料固化，使用由上下吹打冰冷PBS为多次冷却的吸头。 </li><li>把液滴中的阱中一个48井板中的中间和孵化在培养箱中在37℃下进行15分钟固化。 </li><li> 15分钟后，小心地加入250微升补充有类器官培养基的10mM烟酰胺，10μMCHIR99021和10μMY27632。小打乱类器官片段将组建成小囊状类器官在24小时内。 </li></ol>第5天: <ol start="16"><li>刷新介质和选择的抗生素补充（用于嘌呤霉素，使用4微克/毫升）。 </li></ol>第7天: <ol start="17"><li>更换培养基为标准类器官培养液，辅以抗生素的选择。 注意:发芽的将是完整CHIR99021停药后2-3周。选择后，组织体可生长没有选择抗生素。 </li></ol> 4.组织体RNA准备定量RT-PCR和基因芯片<ol><li>用于RNA制备，使用根据制造商的协议的商业试剂盒。从类器官取出中期和直含基质胶类器官的圆顶增加350微升缓冲RLT的，辅以β巯基乙醇。悬浮马泰人在RLT使用P1000微量移液。 </li><li>根据制造商的协议来执行进一步的RNA制备。 </li><li>任选:使用的Ovation微微WTA系统增加的RNA产量通过扩增，根据制造商的协议规定的50微克的总RNA的初始输入。 </li><li>任选:对于前RNA的微阵列的质量控制，使用真核生物总RNA纳米芯片，旨在为8.5以上（ 图5），根据制造商的协议的RNA完整性数（RIN）上的2100生物分析仪上运行样本。 </li></ol> 5.加工类器官的石蜡包埋及免疫组化<ol><li>前类器官嵌入的起始预温热的铝块具有孔配件12毫米直径的玻璃管在培养箱中在70℃下保持液态石蜡。 </li><li>采取单井与完全成熟的组织体，并删除介质，使嵌入式类器官完好。 </li><li>加入1毫升在PBS中的4％多聚甲醛直井和在4℃下的任何地方从1小时至固定过夜。 </li><li>用1ml冰冷的PBS代替多聚甲醛。 任选:在此步骤之后保持固定组织体在PBS中长达1周，在4℃。 </li><li>重悬固定组织体在1ml的PBS中，并放置到玻璃小瓶中。 </li><li>让组织体沉入底部，持续1分钟，滗PBS中并替换，用70％的乙醇，其中一对夫妇的曙红溶液液滴的溶解，使组织体的可视化在整个嵌入处理。 </li><li>留在70％乙醇组织体在室温下。 30分钟后，通过小心地倾析，能够用肉眼来可视化，因为轻微带粉红色的曙红颜色的组织体除去70％的乙醇。更换96％的乙醇嵌入解决方案。 </li><li>重复步骤5.7，每次更换与下一个嵌入解决方案。 70％乙醇，90％乙醇，96％乙醇，100％ETHA:通过以下的溶液随后通过组织体NOL，100％的乙醇，二甲苯，二甲苯等。 </li><li>倒出最后二甲苯洗净，倒入石蜡入管。把该管立即在预加热的铝块，在70℃进行30分钟，并用新的清洁石蜡代替石蜡。 </li><li>使用预温热巴斯德吸管的大开口倾石蜡起飞和吸管组织体入石蜡块模具。保持巴斯德吸管用温暖的本生灯。放置在蜡块模具的所有类器官的一小层液体石蜡。 </li><li>尽可能，尝试操纵使用加热夹层针朝蜡块模具中心的所有组织体。保持解剖针用温暖的本生灯。 </li><li>当组织体在模具中的定位是令人满意的，硬模稍微固化的石蜡层。 </li><li>浇在上面更完成的石蜡块，并添加一个标准组织包埋盒。 </li></ol>

<ol>
	<li>Sato, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+Lgr5+stem+cells+build+crypt-villus+structures+in+vitro+without+a+mesenchymal+niche.">Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche.</a> Nature. 459 (7244), 262-265 (2009).</li><li>Al-Nafussi, A. I., Wright, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+epidermal+growth+factor+(EGF)+on+cell+proliferation+of+the+gastrointestinal+mucosa+in+rodents.">The effect of epidermal growth factor (EGF) on cell proliferation of the gastrointestinal mucosa in rodents.</a> Virchows Archiv. B, Cell Pathology Including Molecular Pathology. 40 (1), 63-69 (1982).</li><li>Haramis, A. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=De+novo+crypt+formation+and+juvenile+polyposis+on+BMP+inhibition+in+mouse+intestine.">De novo crypt formation and juvenile polyposis on BMP inhibition in mouse intestine.</a> Science. 303 (5664), 1684-1686 (2004).</li><li>Zhao, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=R-spondin1,+a+novel+intestinotrophic+mitogen,+ameliorates+experimental+colitis+in+mice.">R-spondin1, a novel intestinotrophic mitogen, ameliorates experimental colitis in mice.</a> Gastroenterology. 132 (4), 1331-1343 (2007).</li><li>Schwank, G., Andersson-Rolf, A., Koo, B. K., Sasaki, N., Clevers, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+BAC+transgenic+epithelial+organoids.">Generation of BAC transgenic epithelial organoids.</a> PLoS One. 8 (10), e76871 (2013).</li><li>Koo, B. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+gene+expression+in+primary+Lgr5+organoid+cultures.">Controlled gene expression in primary Lgr5 organoid cultures.</a> Nature Methods. 9 (1), 81-83 (2012).</li><li>Heijmans, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ER+stress+causes+rapid+loss+of+intestinal+epithelial+stemness+through+activation+of+the+unfolded+protein+response.">ER stress causes rapid loss of intestinal epithelial stemness through activation of the unfolded protein response.</a> Cell Rep. 3 (4), 1128-1139 (2013).</li><li>Roe, T., Reynolds, T. C., Yu, G., Brown, P. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integration+of+murine+leukemia+virus+DNA+depends+on+mitosis.">Integration of murine leukemia virus DNA depends on mitosis.</a> The EMBO Journal. 12 (5), 2099-2108 (1993).</li><li>Lau, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lgr5+homologues+associate+with+Wnt+receptors+and+mediate+R-spondin+signalling.">Lgr5 homologues associate with Wnt receptors and mediate R-spondin signalling.</a> Nature. 476 (7360), 293-297 (2011).</li><li>Bahnson, A. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Centrifugal+enhancement+of+retroviral+mediated+gene+transfer.">Centrifugal enhancement of retroviral mediated gene transfer.</a> Journal Of Virological Methods. 54 (2-3), 131-143 (1995).</li></ol>

The authors have nothing to disclose.

目前的视频协议描述了从初级肠上皮而这些类器官采用定量RNA技术和免疫组化下游分析组织体的慢病毒转导。 往往是在贴壁或悬浮细胞在培养板中进行慢病毒转导。自组织体的三维结构使得它们难以通过病毒颗粒渗透，使用了若干方法来增加疗效。转导后使用CHIR99021组织体的预处理增加增殖，从而可行性。重要的是要保持小细胞群体胰蛋白酶消化后，由于单电池已减少存活?...

肠上皮细胞是最迅速增殖的身体组织，这导致它吸引了来自研究癌症和干细胞广泛兴趣之一。在2009年的技术被发布到基质胶产生的小肠隐窝持久的文化，涵养一个三维结构1。这些结构中，称为肠组织体，可以使用标准技术进行培养，与周围的介质补充有若干所定义的生长因子，包括BMP-信号通路抑制剂头发生素（木栓）中，Wnt信号通路的增强rspondin 1（Rspo1）和表皮生长因子（EGF）的所有发现增强肠道增殖2-4。 类器官超越了传统方面的癌细胞系，他们都是非突变，保持干细胞的层次结构，显示完整的细胞极化，表现出分化成新生的小INT发现的所有细胞系estinal上皮。因为它们可被转导携带的转基因或RNA干扰构造5，它们被用于研究特定的遗传元件，使用转基因小鼠在成本和速度的面抵销实验。就可以利用小鼠逆转录病毒或慢病毒载体6,7进行转基因表达的组织体。由于鼠逆转录病毒，能够转导分裂细胞只8的局限性，慢病毒转导是更频繁地用于细胞，很难感染，如组织体。 病毒转导和稳定表达的转基因组织体可用于多种下游分析，包括定量的RNA分析和免疫组织化学。综合来看，从小学肠上皮细胞组织体的文化已经演变成一个常规技术，很容易没有具体的实验室要求实施，并已成为CE小说标准LL文化对肠道上皮细胞的研究。 病毒转导和组织体随后的下游分析技术是繁琐的执行，并帮助组织体实验，我们产生这个视频协议，显示培养的组织体的慢病毒转导的方法。我们还展示了如何组织体正确处理可以提高产量，因此利用RNA技术或免疫增强下游的分析性能。在协议中，从它们的小肠隐窝中衍生的组织体被单独使用，但所描述的技术可应用于结肠组织体为好。

<table border="0" cellpadding="0" cellspacing="0" width="539">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:345px;">Reagent</td>
			<td style="width:91px;">Company&#160;</td>
			<td style="width:103px;">Cat. No.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:345px;">Polyethylene imine</td>
			<td style="width:91px;">polysciences</td>
			<td style="width:103px;">23966-2</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:345px;">DMEM medium</td>
			<td style="width:91px;">Lonza</td>
			<td style="width:103px;">BE12-614F</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:345px;">Fetal calf serum</td>
			<td style="width:91px;">Lonza</td>
			<td style="width:103px;">DE14-801F</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:345px;">Penicillin-streptomycin</td>
			<td style="width:91px;">Invitrogen</td>
			<td style="width:103px;">15140-122</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:345px;">Glutamin</td>
			<td style="width:91px;">Invitrogen</td>
			<td style="width:103px;">25030-024</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:345px;">matrigel</td>
			<td style="width:91px;">BD</td>
			<td style="width:103px;">BD 356231</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:345px;">Advanced DMEM-F12</td>
			<td style="width:91px;">Gibco</td>
			<td style="width:103px;">12634-010</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:345px;">N2</td>
			<td style="width:91px;">Invitrogen</td>
			<td style="width:103px;">17502-048</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:345px;">B27</td>
			<td style="width:91px;">Invitrogen</td>
			<td style="width:103px;">17504-044</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:345px;">N-acetyl cysteine</td>
			<td>Sigma</td>
			<td>A9165-1G</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">mouse Egf</td>
			<td>Invitrogen</td>
			<td>PMG8045</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hepes 1M</td>
			<td>Invitrogen</td>
			<td>15630-056</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">glutamax 100x</td>
			<td>Invitrogen</td>
			<td>35050-038</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Chir 99021</td>
			<td>axon</td>
			<td>1386</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Y27632&#160;</td>
			<td>Sigma</td>
			<td>Y0503-5MG</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">polybrene</td>
			<td>Sigma</td>
			<td>107689</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">nicotinamide</td>
			<td>Sigma</td>
			<td>N0636</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trypsin</td>
			<td>Lonza</td>
			<td>BE02-007E</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">puromycin</td>
			<td>sigma</td>
			<td>P 7255</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Rneasy mini kit</td>
			<td>Qiagen</td>
			<td>74106</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">b-mercaptoethanol</td>
			<td>Merck</td>
			<td>8,057,400,250</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ovation Pico WTA system</td>
			<td>NuGen</td>
			<td>3300-12</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">paraformaldehyde</td>
			<td>Sigma</td>
			<td>252549-1L</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">glass vial conical 12mm x 75mm 5ml</td>
			<td>VWR</td>
			<td>LSUKM12</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Eosin Yellowish</td>
			<td>VWR</td>
			<td>1,159,350,025</td>
		</tr>
	</tbody>
</table>

a protocol for lentiviral transduction and downstream analysis of intestinal organoids

Intestinal crypt-villus structures termed organoids, can be kept in sustained culture three dimensionally when supplemented with the appropriate growth factors. Since organoids are highly similar to the original tissue in terms of homeostatic stem cell differentiation, cell polarity and presence of all terminally differentiated cell types known to the adult intestinal epithelium, they serve as an essential resource in experimental research on the epithelium. The possibility to express transgenes or interfering RNA using lentiviral or retroviral vectors in organoids has increased opportunities for functional analysis of the intestinal epithelium and intestinal stem cells, surpassing traditional mouse transgenics in speed and cost. In the current video protocol we show how to utilize transduction of small intestinal organoids with lentiviral vectors illustrated by use of doxycylin inducible transgenes, or IPTG inducible short hairpin RNA for overexpression or gene knockdown. Furthermore, considering organoid culture yields minute cell counts that may even be reduced by experimental treatment, we explain how to process organoids for downstream analysis aimed at quantitative RT-PCR, RNA-microarray and immunohistochemistry. Techniques that enable transgene expression and gene knock down in intestinal organoids contribute to the research potential that these intestinal epithelial structures hold, establishing organoid culture as a new standard in cell culture.

类器官慢病毒转导 利用慢病毒颗粒类器官传导技术事先和传导过程依赖于正确处理组织体。组织体（ 图3A）进行培养，它们被破碎成单个隐窝（ 图3B）。如以前报道的，这些单隐窝，当GSK3抑制剂CHIR99021的存在下培养成为囊性隐窝9（ 图3C）。随后组织体进行胰蛋白酶处理，以允许病毒颗粒的单细胞的渗透。当转导细胞的慢...

Watch this Scientific Journal Video about A Protocol for Lentiviral Transduction and Downstream Analysis of Intestinal Organoids at JoVE.com

A Protocol for Lentiviral Transduction and Downstream Analysis of Intestinal Organoids

In this video protocol we give a step by step explanation of lentiviral transduction in organoids of primary intestinal epithelium and of processing and downstream analysis of these cultures by quantitative RT-PCR, RNA-microarray and immunohistochemistry.

协议的慢病毒转导与肠组织体下游分析

Intestinal crypt-villus structures termed organoids, can be kept in sustained culture three dimensionally when supplemented with the appropriate growth ...

a-protocol-for-lentiviral-transduction-downstream-analysis-intestinal

Research

JoVE Journal

Biology

30.4K 次观看.  Academical Medical Center. In this video protocol we give a step by step explanation of lentiviral transduction in organoids of primary intestinal epithelium and of processing and downstream analysis of these cultures by quantitative RT-PCR, RNA-microarray and immunohistochemistry. 

视频: A Protocol for Lentiviral Transduction and Downstream Analysis of Intestinal Organoids

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 ...

科研

生物学

Detection of Protein Ubiquitination

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of three enzymes. They include ubiquitin-activating enzyme E1, ubiquitin-conjugating enzyme E2, and ubiquitin ligase E3. Unlike to E1 and E2, E3 ubiquitin ligases display substrate specificity. On the other hand, numerous deubiquitylating enzymes have roles in processing polyubiquitinated proteins. Ubiquitination can result in change of protein stability, cellular localization, and biological activity. Mutations of genes involved in the ubiquitination/deubiquitination pathway or altered ubiquitin system function are associated with many different human diseases such as various types of cancer, neurodegeneration, and metabolic disorders. The detection of altered or normal ubiquitination of target proteins may provide a better understanding on the pathogenesis of these diseases. &nbsp;Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro. These protocols are also useful to detect other ubiquitin-like small molecule modification such as sumolyation and neddylation.

Ubiquitination is a key posttranslational modification carried out by a set of three enzymes. Mutations of genes involved in this modification are associated with many different human diseases. Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro.

蛋白泛素化的检测

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of ...

Generation of Induced Pluripotent Stem Cells by Reprogramming Mouse Embryonic Fibroblasts with a Four Transcription Factor, Doxycycline Inducible Lentiviral Transduction System

Using a defined set of transcription factors and cell culture conditions, Yamanaka and colleagues demonstrated that retrovirus-mediated delivery and expression of Oct4, Sox2, c-Myc, and Klf4 is capable of inducing pluripotency in mouse fibroblasts.1 Subsequent reports have demonstrated the utility of the doxycycline (DOX) inducible lentiviral delivery system for the generation of both primary and secondary iPS cells from a variety of other adult mouse somatic cell types.2,3
Induced pluripotent stem (iPS) cells are similar to embryonic stem (ES) cells in morphology, proliferation and ability to induce teratoma formation. Both types of cell can be used as the pluripotent starting material for the generation of differentiated cells or tissues in regenerative medicine.4-6 iPS cells also have a distinct advantage over ES cells as they exhibit key properties of ES cells without the ethical dilemma of embryo destruction.
Here we demonstrate the protocol for reprogramming mouse embryonic fibroblast (MEF) cells with the Stemgent DOX Inducible Mouse TF Lentivirus Set. We also demonstrate that the Stemgent DOX Inducible Mouse TF Lentivirus Set is capable of expressing each of the four transcription factors upon transduction into MEFs thereby inducing a pluripotent stem cell state that displays the pluripotency markers characteristic of ES cells.

The Stemgent Dox Inducible Mouse TF Lentivirus Set can reprogram mouse embryonic fibroblasts (MEFs) to induced pluripotent stem (iPS) cells. Here we demonstrate the protocol for DOX-inducible expression of mouse reprogramming transcription factors Oct4, Sox2, Klf4 and c-Myc to generate iPS colonies that express common mES pluripotency markers.

重新用四个转录因子的小鼠胚胎成纤维细胞诱导多能干细胞的产生，强力霉素诱导慢病毒转导系统

Using a defined set of transcription factors and cell culture conditions, Yamanaka and colleagues demonstrated that retrovirus-mediated delivery and ...

Proteomic Sample Preparation from Formalin Fixed and Paraffin Embedded Tissue

Preserved clinical material is a unique source for proteomic investigation of human disorders. Here we describe an optimized protocol allowing large scale quantitative analysis of formalin fixed and paraffin embedded (FFPE) tissue. The procedure comprises four distinct steps. The first one is the preparation of sections from the FFPE material and microdissection of cells of interest. In the second step the isolated cells are lysed and processed using 'filter aided sample preparation' (FASP) technique. In this step, proteins are depleted from reagents used for the sample lysis and are digested in two-steps using endoproteinase LysC and trypsin. 
After each digestion, the peptides are collected in separate fractions and their content is determined using a highly sensitive fluorescence measurement. Finally, the peptides are fractionated on 'pipette-tip' microcolumns. The LysC-peptides are separated into 4 fractions whereas the tryptic peptides are separated into 2 fractions. In this way prepared samples allow analysis of proteomes from minute amounts of material to a depth of 10,000 proteins. Thus, the described workflow is a powerful technique for studying diseases in a system-wide-fashion as well as for identification of potential biomarkers and drug targets.

Archival formalin fixed and paraffin embedded (FFPE) clinical samples are valuable material for investigation of diseases. Here we demonstrate a sample preparation workflow allowing in-depth proteomic analysis of microdissected FFPE tissue.

修正了福尔马林蛋白质组学样品的制备和石蜡包埋组织

Preserved clinical material is a unique source for proteomic investigation of human disorders. Here we describe an optimized protocol allowing large scale ...

Live Cell Imaging of Primary Rat Neonatal Cardiomyocytes Following Adenoviral and Lentiviral Transduction Using Confocal Spinning Disk Microscopy

Primary rat neonatal cardiomyocytes are useful in basic in vitro cardiovascular research because they can be easily isolated in large numbers in a single procedure. Due to advances in microscope technology it is relatively easy to capture live cell images for the purpose of investigating cellular events in real time with minimal concern regarding phototoxicity to the cells. This protocol describes how to take live cell timelapse images of primary rat neonatal cardiomyocytes using a confocal spinning disk microscope following lentiviral and adenoviral transduction to modulate properties of the cell. The application of two different types of viruses makes it easier to achieve an appropriate transduction rate and expression levels for two different genes. Well focused live cell images can be obtained using the microscope&#8217;s autofocus system, which maintains stable focus for long time periods. Applying this method, the functions of exogenously engineered proteins expressed in cultured primary cells can be analyzed. Additionally, this system can be used to examine the functions of genes through the use of siRNAs as well as of chemical modulators.

This protocol describes a method of live cell imaging using primary rat neonatal cardiomyocytes following lentiviral and adenoviral transduction using confocal spinning disk microscopy. This enables detailed observations of cellular processes in living cardiomyocytes.

原代大鼠乳鼠心肌细胞的活细胞成像继腺病毒和慢病毒转导利用共聚焦旋转盘显微镜

Primary rat neonatal cardiomyocytes are useful in basic in vitro cardiovascular research because they can be easily isolated in large numbers in a single ...

A Video Protocol of Retroviral Infection in Primary Intestinal Organoid Culture

Lgr5-positive stem cells can be supplemented with the essential growth factors Egf, Noggin, and R-Spondin, which allows us to culture ever-expanding primary 3D epithelial structures in vitro. Both the architecture and physiological properties of these &#39;mini-guts&#39;, also called organoids, closely resemble their in vivo counterparts. This makes them an attractive model system for the small intestinal epithelium. Using retroviral transduction, functional genetics can now be performed by conditional gene overexpression or knockdown. This video demonstrates the procedure of organoid culture, the generation of retroviruses, and the retroviral transduction of organoids to assist phenotypic analysis of the small intestinal epithelium in vitro. This novel organotypic model system in combination with retroviral mediated gene expression provides a valuable tool for rapid analysis of gene function in vitro without the need of costly and time-consuming generation for transgenic animals.

This protocol explains primary Lgr5-positve organoid culture and the subsequent performance of retroviral transduction. This enables Cre-inducible overexpression or knockdown of the delivered transgene and allows functional studies to be carried out in the novel in vitro organotypic model system.

逆转录病毒感染的原发性肠化培养的视频协议

Lgr5-positive stem cells can be supplemented with the essential growth factors Egf, Noggin, and R-Spondin, which allows us to culture ever-expanding ...

Clock Scan Protocol for Image Analysis: ImageJ Plugins

The clock scan protocol for image analysis is an efficient tool to quantify the average pixel intensity within, at the border, and outside (background) a closed or segmented convex-shaped region of interest, leading to the generation of an averaged integral radial pixel-intensity profile. This protocol was originally developed in 2006, as a visual basic 6 script, but as such, it had limited distribution. To address this problem and to join similar recent efforts by others, we converted the original clock scan protocol code into two Java-based plugins compatible with NIH-sponsored and freely available image analysis programs like ImageJ or Fiji ImageJ. Furthermore, these plugins have several new functions, further expanding the range of capabilities of the original protocol, such as analysis of multiple regions of interest and image stacks. The latter feature of the program is especially useful in applications in which it is important to determine changes related to time and location. Thus, the clock scan analysis of stacks of biological images may potentially be applied to spreading of Na+ or Ca++ within a single cell, as well as to the analysis of spreading activity (e.g., Ca++ waves) in populations of synaptically-connected or gap junction-coupled cells. Here, we describe these new clock scan plugins and show some examples of their applications in image analysis.

This paper describes two novel ImageJ plugins for &#39;Clock Scan&#39; image analysis. These plugins expand the functionality of the original visual basic 6 program and, most importantly, make the program available to a large research community by bundling it with the ImageJ free image analysis software package.

用于图像分析的时钟扫描协议:ImageJ插件

The clock scan protocol for image analysis is an efficient tool to quantify the average pixel intensity within, at the border, and outside (background) a ...

Laboratory Protocol for Genetic Gut Content Analyses of Aquatic Macroinvertebrates Using Group-specific rDNA Primers

Analyzing food webs is essential for a better understanding of ecosystems. For example, food web interactions can undergo severe changes caused by the invasion of non-indigenous species. However, an exact identification of field predator-prey interactions is difficult in many cases. These analyses are often based on a visual evaluation of gut content or the analysis of stable isotope ratios (&#948;15N and &#948;13C). Such methods require comprehensive knowledge about, respectively, morphologic diversity or isotopic signature from individual prey organisms, leading to obstacles in the exact identification of prey organisms. Visual gut content analyses especially underestimate soft bodied prey organisms, because maceration, ingestion and digestion of prey organisms make identification of specific species difficult. Hence, polymerase chain reaction (PCR) based strategies, for example the use of group-specific primer sets, provide a powerful tool for the investigation of food web interactions. Here, we describe detailed protocols to investigate the gut contents of macroinvertebrate consumers from the field using group-specific primer sets for nuclear ribosomal deoxyribonucleic acid (rDNA). DNA can be extracted either from whole specimens (in the case of small taxa) or out of gut contents of specimens collected in the field. Presence and functional efficiency of the DNA templates need to be confirmed directly from the tested individual using universal primer sets targeting the respective subunit of DNA. We also demonstrate that consumed prey can be determined further down to species level via PCR with unmodified group-specific primers combined with subsequent single strand conformation polymorphism (SSCP) analyses using polyacrylamide gels. Furthermore, we show that the use of different fluorescent dyes as labels enables parallel screening for DNA fragments of different prey groups from multiple gut content samples via automated fragment analysis.

Most common gut content analyses of macroinvertebrates are visual. Requiring intense knowledge about morphological diversity of prey organisms, they miss soft bodied prey and, due to strong comminution of prey, are nearly impossible for some organisms, including amphipods. We provide detailed, novel genetic approaches for macroinvertebrate prey identification in the diet of amphipods.

用基团特异 rDNA 引物对水生无脊椎动物进行遗传肠道含量分析的实验室规程

Analyzing food webs is essential for a better understanding of ecosystems. For example, food web interactions can undergo severe changes caused by the ...

Laser-assisted Lentiviral Gene Delivery to Mouse Fertilized Eggs

Lentiviruses are efficient vectors for gene delivery to mammalian cells. Following transduction, the lentiviral genome is stably incorporated into the host chromosome and is passed on to progeny. Thus, they are ideal vectors for creation of stable cell lines, in vivo delivery of indicators, and transduction of single cell fertilized eggs to create transgenic animals. However, mouse fertilized eggs and early stage embryos are protected by the zona pellucida, a glycoprotein matrix that forms a barrier against lentiviral gene delivery. Lentiviruses are too large to penetrate the zona and are typically delivered by microinjection of viral particles into the perivitelline cavity, the space between the zona and the embryonic cells. The requirement for highly skilled technologists and specialized equipment has minimized the use of lentiviruses for gene delivery to mouse embryos. This article describes a protocol for permeabilizing the mouse fertilized eggs by perforating the zona with a laser. Laser-perforation does not result in any damage to embryos and allows lentiviruses to gain access to embryonic cells for gene delivery. Transduced embryos can develop into blastocyst in vitro, and if implanted in pseudopregnant mice, develop into transgenic pups. The laser used in this protocol is effective and easy to use. Genes delivered by lentiviruses stably incorporate into mouse embryonic cells and are germline transmittable. This is an alternative method for creation of transgenic mice that requires no micromanipulation and microinjection of fertilized eggs.

Mouse fertilized eggs and early stage embryos are protected by the zona pellucida, a glycoprotein matrix that forms a barrier against gene delivery. This article describes a protocol for perforating the zona with a laser to transduce embryonic cells with lentiviral vectors and to create transgenic mice.

激光辅助慢病毒基因传递到小鼠受精卵

Lentiviruses are efficient vectors for gene delivery to mammalian cells. Following transduction, the lentiviral genome is stably incorporated into the ...

3D Culturing of Organoids from the Intestinal Villi Epithelium Undergoing Dedifferentiation

Clonogenicity of organoids from the intestinal epithelium is attributed to the presence of stem cells therein. The mouse small intestinal epithelium is compartmentalized into crypts and villi: the stem and proliferating cells are confined to the crypts, whereas the villi epithelium contains only differentiated cells. Hence, the normal intestinal crypts, but not the villi, can give rise to organoids in 3D cultures. The procedure described here is applicable only to villus epithelium undergoing dedifferentiation leading to stemness. The method described uses the Smad4-loss-of-function:&#946;-catenin gain-of-function (Smad4KO:&#946;-cateninGOF) conditional mutant mouse. The mutation causes the intestinal villi to dedifferentiate and generate stem cells in the villi. Intestinal villi undergoing dedifferentiation are scraped off the intestine using glass slides, placed in a 70 &#181;m strainer and washed several times to filter out any loose cells or crypts prior to plating in BME-R1 matrix to determine their organoid-forming potential. Two main criteria were used to ensure that the resulting organoids were developed from the dedifferentiating villus compartment and not from the crypts: 1) microscopically evaluating&#160;the isolated villi to ensure absence of any tethered crypts, both before and after plating in the 3D matrix, and 2) monitoring the time course of organoid development from the villi. Organoid initiation from the villi occurs only two to five days after plating and appears irregularly shaped, whereas the crypt-derived organoids from the same intestinal epithelium are apparent within sixteen hours of plating and appear spherical. The limitation of the method, however, is that the number of organoids formed, and the time required for organoid initiation from the villi vary depending on the degree of dedifferentiation. Hence, depending upon the specificity of the mutation or the insult causing the dedifferentiation, the optimal stage at which villi can be harvested to assay their organoid forming potential, must be determined empirically.

The procedure describes isolation of the villi from the mouse intestinal epithelium undergoing dedifferentiation to determine their organoid forming potential.

肠道维利表皮的器官3D培养进行分化

Clonogenicity of organoids from the intestinal epithelium is attributed to the presence of stem cells therein. The mouse small intestinal epithelium is ...