Name: methods to classify cytoplasmic foci as mammalian stress granules
Uploaded: 2017-05-12T14:00:00
Description: Watch this Scientific Journal Video about Methods to Classify Cytoplasmic Foci as Mammalian Stress Granules at JoVE.com

我们感谢Ivanov和Anderson实验室的成员对这份手稿的有益的讨论和反馈。这项工作得到了国家卫生研究院（GM111700和CA168872至PA，NS094918至PI），波兰国家科学中心[授予UMO-2012/06 / M / NZ3 / 00054至WS]的支持。 WS还承认波兰的科学和高等教育部（Mobility Plus计划）和波兰美国富布莱特委员会在美国的研究经费支持。

细胞准备<ol><li>向24孔板的12个孔中加入高压灭菌的盖玻片， 如图1A所示 ;这可以使用连接到真空的无菌巴斯德吸管来接收每个盖玻片。轻轻点击井的盖玻片释放吸力，允许盖玻片落入井中。 </li><li>每孔1×10 5 U-2 OS（U2OS）骨肉瘤细胞，最终体积为500μL培养基。将板一侧移动到一边上下几次，以确保细胞均匀分布。 </li><li>用清洁的P1000小头轻轻按下盖玻片，以确保盖玻片位于井?...

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(87), (2014).</li><li>Wippich, F., 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=Dual+specificity+kinase+DYRK3+couples+stress+granule+condensation/dissolution+to+mTORC1+signaling.">Dual specificity kinase DYRK3 couples stress granule condensation/dissolution to mTORC1 signaling.</a> Cell. 152 (4), 791-805 (2013).</li></ol>

如通过多种疾病的免疫组织学研究证明，慢性应激导致不同细胞内病灶的形成。例如，大多数神经变性疾病的特征在于不溶性蛋白质的细胞内聚集体。 SG相关蛋白在这种聚集体中的存在通常是得出这样的焦点是SG的基础。在用新的应激刺激处理的细胞中观察到SG标记阳性细胞质病灶时也得出了类似的结论。该协议提供了一个简单的工作流程来识别真正的 SG。 推定的SGs必?...

细胞采用许多机制来应对压力。一些反应发生在转录后水平，并涉及调节mRNA翻译和/或稳定性1,2 。应激调节的mRNA翻译阻滞和降解与特异性非膜细胞病灶的形成相关，其最好的特征是应激颗粒（SGs​​） 3 。 SG是细胞质焦点，其将非翻译的mRNP浓缩在对胁迫（ 例如，氧化，热休克，营养物饥饿和病毒感染）反应的缓冲细胞中。除了非翻译mRNA之外，SG还含有翻译起始因子，RNA结合蛋白和各种信号转导蛋白。 SG是抑制蛋白质翻译和RNA代谢改变的生物标志物，并与细胞存活和细胞凋亡信号通路有关和核工程5 。 SG是动态实体，它们的形成与细胞翻译的状态紧密相关6 。尽管它们看起来很稳固，但大多数SG蛋白质组件都能快速穿梭进出，停留时间为秒。虽然SG持续了几分钟到几个小时，但其大多数组件都处于快速通量。翻译起始的抑制和随后的翻译多核糖体的拆卸促进了SG的形成;因此，SGs与翻译多核糖体处于平衡状态。这种多聚体/ SG平衡是将真正的 SG与其他应激诱发的病灶区分开来的关键6,7 。 抑制翻译起始需要转录含有mRNA，翻译起始因子（eIF）和40S核糖体亚基（s）的翻译能力的起始前复合物<html>称为48S复合物）转化为可以聚结成SG 4,6的翻译失活复合物（所谓的48S *复合物）。 SGs通过在48S复合物形成上游的两个不同步骤的翻译阻滞来促进：干扰cap结合eIF4F复合物的功能（ 例如靶向其eIF4A亚基）或翻译起始因子eIF2的α亚基的磷酸化，由一个介导的或更多的四种eIF2激酶。停滞的48S复合物（ 即 40S核糖体亚基和选择的翻译起始因子）的存在是SG8,9的标志。 SG已经与各种病理状态相关联，例如病毒感染，神经变性，自身免疫和癌症3,10,11 ，ss =“xref”&gt; 12，13。几种SG成分的突变形式与神经变性疾病（ 例如，肌萎缩性侧索硬化症）相关，其中神经元显示可能在神经元死亡中起主要作用的细胞内病理性包涵体13 。一些病毒劫持SG成分以抑制SG形成和增强病毒复制14 。最近的研究还将SG与癌症15联系起来，并将化疗和放疗治疗的癌细胞存活10,16,17,18,19,20。虽然这些发现激发了对SG生物学的极大兴趣，但许多已发表的报告缺乏重要的控制来区分真正的 SG的形成和其他压力引起的病灶。 SG最初描述为由选定的mRNA结合蛋白（RBP）组成的非膜性细胞质病灶，包括T细胞细胞抗原1（TIA-1）和Poly-A结合蛋白（PABP）;翻译起始因素;聚腺苷酸化mRNA;和小核糖体亚基4,6,21,22。传统上，它们的组成通过诸如免疫染色和荧光标记蛋白的异位表达的技术23,24来确定。由于其高度动态的性质，SG蛋白和/或mRNA的免疫定位仍然是检测SG 23,24的定义方法。迄今为止，许多RBP和其他蛋白质（超过120种不同的蛋白质）被描述为SG组分11 。多少SG-局部蛋白质以应激依赖性和独立性方式改变其定位，并且可以在其他细胞内区室和灶点积累，选择正确的SG标记物并采用功能标准来区分SG与其他类型的应激诱导灶。 真正的 SGs含有mRNA，翻译起始因子和小核糖体亚基，并且与翻译处于动态平衡。 这个简单的工作流程旨在确定应激诱发的病灶是否真正的 SGs。该工作流程包括使用U2OS细胞的几种实验方法，通常用于研究SG的细胞。这些细胞是理想的，因为它们具有大的细胞质，相对平坦，并且牢固地附着在玻璃盖玻片上。其他细胞类型可用于研究SG，但重要的是要注意，时间，药物浓度和SG-成核蛋白丰度的差异可以改变动力学和组合sition。另外，一些细胞通过形成细胞表面泡泡来响应多聚甲醛固定，然后产生引起一些SG标记物的点状定位的皱纹外观;没有仔细分析，这些可以被分类为SG。这突出了必须评估将应激诱发的病灶识别为真正的 SG所需的所有标准。长春瑞滨（VRB）是一种促进SG 20的癌症治疗剂，以及砷酸钠（SA），一种健壮且性能良好的SG诱导剂，被用作本协议实验的应力。 标准SGs包含多个SG标记（蛋白质和mRNA），共定位于细胞质病灶。该方案同时使用免疫荧光和荧光原位杂交（FISH）检测蛋白质标记和聚腺苷酸化mRNA 22的定位。简言之，间接免疫荧光使用抗体（即即一级抗体）对于给定的蛋白质是特异的，以在细胞内检测它。然后，附着于荧光染料的二抗（通常是物种特异性）识别第一抗体并揭示靶蛋白定位。荧光显微镜用于检测细胞内的局部信号。使用不同物种产生的抗体并用不同颜色的二级抗体进行检测，可以检测多种抗体的共定位，表明它们的蛋白质靶标位于同一位置23 。选择已被验证以在真正的 SG上共定位的标记是重要的。 FISH使用与特定RNA或DNA序列碱基对的标记探针25 。为了检测mRNA，该方案使用与mRNA的polyA尾杂交（或碱基对）的生物素化寡核苷酸（dT） 40探针（ 即 polyA FISH）。然后使用荧光偶联的链霉亲和素检测生物素化的探针，因为链霉亲和素对生物素具有高亲和力。当评估SG时，重要的是将polyA FISH与免疫荧光检测SG标记物结合，如真正的 SG，两个信号应共定位。 使用该协议，共定位以多种方式进行评估。在多通道（ 即 RGB：红色，绿色和蓝色）图像中，colocalization将改变重叠信号的颜色（ 例如， colocalized红色和绿色出现黄色） 23 。另外，使用线扫描分析图形化定量共定位，其中在给定线8,20上测量每个颜色的强度。该协议描述了使用ImageJ 26的两行扫描分析程序。一个程序是手动的，经过整个过程，另一个使用宏，或者一个自动执行手动步骤的简单程序。重要的是要通过手动程序来了解宏程序。 SGs形成在翻译被压制的单元格中;因此，与未处理的细胞相比，具有SG的细胞应该显示出降低的全局翻译水平。实验上，使用了尿布溴霉素。在固定之前，将嘌呤霉素和埃马汀加入细胞持续一段时间，使嘌呤霉素掺入活跃形成的多肽中，导致终止27,28 。需要用emetine进行治疗以防止再次起搏29 。然后可以使用抗嘌呤霉素抗体检测嘌呤霉素，给出活性翻译的快照。使用这种方法是因为它很快，不需要用缺乏特定氨基酸的介质（并且潜在地预处理细胞）的预饥饿，并且揭示了蛋白质翻译的亚细胞定位。已经使用其他方法，特别是使用修饰的氨基酸类似物，例如甲硫氨酸类似物L-叠氮基低聚维生素（AHA），与“click-it”化学30相结合 ，表明含有SG的细胞显示比相邻细胞31低得多的翻译。然而，该技术需要甲硫氨酸饥饿，然后脉冲标记15-30分钟。蛋氨酸饥饿构成额外的压力，而长的标签时间（ 即 15-30分钟）导致测量累积而不是正在进行的翻译，并且还允许新合成的蛋白质从其合成位点移动到其最终目的地内细胞。相比之下，番红霉素更快，并且与任何培养基（例如用于通过葡萄糖饥饿诱导SG的无葡萄糖培养基）相容。 规范SGs处于动态平衡状态积极翻译多核糖体。这可以通过用稳定或不稳定多核糖体的药物处理样品来实验地评估，从而在SG和多核糖体之间平衡23 。通过将核糖体“冻结”到mRNA上，环己酰亚胺（或其类似的表现）阻断延伸，从而减少能够形成SG的可用非多核糖体mRNP的库。实验上可以通过以下两种方法使用：在应力之前加入放线菌酮（或埃米汀）以防止SG形成，或者在形成SG之后加入放线菌酮（或埃米汀），即使没有除去应激剂，也会导致SG拆卸停止早产复合物缓慢进入多聚糖部分。相比之下，嘌呤霉素引起过早终止并促进多聚体拆解，增加能够组装成SG的起始mRNA的集合。实验上，嘌呤霉素治疗会增加SG的数量或降低threshold，其响应于分级应激或药物剂量形成。当评估嘌呤霉素的作用时，重要的是使用目的药物的亚最大水平，因为嘌呤霉素预期会增加药物的作用并增加显示SGs的细胞的百分比 - 如果药物不能发生，则不会发生/压力最初导致100％细胞中的SG。这与放线菌酮处理形成对照，当约95％的细胞初始显示SG时，分解SG并且最好地工作，以便可以观察到并且准确地量化最大可能的降低。 该协议提供了研究哺乳动物细胞中SGs的框架。方法包括：（1）免疫荧光染色和ImageJ分析，评估推定的SGs中经典SG相关标记eIF4G，eIF3b和Ras GTPase激活蛋白结合蛋白1（G3BP1）的共定位; （2）寡聚（dT）荧光原位杂交（polyA FISH）检测多聚腺苷酸化mRNA; （3）放线菌酮和嘌呤霉素处理，以确定应激诱导的病灶是否与多核糖体处于动态平衡状态;和（4）番红霉素以评估含有推定的SG的细胞的翻译状态。在一起，这些测定法可以确定应激诱导的病灶是否可归类为真正的 SG。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>U-2 OS</td>
			<td>ATCC</td>
			<td>ATCC&#174;HTB-96&#8482;</td>
			<td>- commonly written as &#34;U2OS&#34; 
			- culture at 37 &#176;C under 5% CO2 in 10% FBS/DMEM</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modification of Eagle&#39;s Medium</td>
			<td>Corning</td>
			<td>10-013-CV</td>
			<td>- abbreviated DMEM 
			- contains 4.5 g/L glucose, pyruvate, and phenol red 
			- supplemented with 10% FBS, 10 mM HEPES, and penicillin/steptomycin 
			- prewarm prior drug(s) dilution</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Sigma</td>
			<td>F2442-500ml</td>
			<td>- abbreviated FBS 
			- used to supplement media</td>
		</tr>
		<tr>
			<td>HEPES (1 M)</td>
			<td>Thermo Fisher Scientific</td>
			<td>15630-080</td>
			<td>- used to supplement media</td>
		</tr>
		<tr>
			<td>penicilline streptomycine</td>
			<td>Sigma</td>
			<td>P0781-100ml</td>
			<td>- used to supplement media</td>
		</tr>
		<tr>
			<td>24 well plate</td>
			<td>Costar</td>
			<td>3524</td>
			<td></td>
		</tr>
		<tr>
			<td>lab tissues</td>
			<td>KIMTECH SCIENCE</td>
			<td>34120</td>
			<td>- commonly called &#34;kimwipes&#34;</td>
		</tr>
		<tr>
			<td>Coverglass for Growth Cover Glasses</td>
			<td>Fisher Scientific</td>
			<td>12-545-82</td>
			<td>- autoclaved before used 
			- keep sterile in the tissue culture hood</td>
		</tr>
		<tr>
			<td>Sodium (meta)arsenite &#8805;90%</td>
			<td>Sigma</td>
			<td>S7400-100G</td>
			<td>- commonly called sodium arsenite and abbreviated SA 
			- dissolution in water at 1 M concentration, then dilute to 100 &#181;M as a working stock</td>
		</tr>
		<tr>
			<td>Vinorelbine</td>
			<td>TSZ CHEM</td>
			<td>RS055</td>
			<td>- abbreviated VRB 
			- dissolve in water to 10 mM</td>
		</tr>
		<tr>
			<td>Puromycin</td>
			<td>Sigma</td>
			<td>P9620</td>
			<td>- used to assess translation level (ribopuromycylation) 
			- used to assess SG connection with active translation 
			- dilute in water to 10 mg/mL</td>
		</tr>
		<tr>
			<td>Emetine dihydrochloride hydrate</td>
			<td>Sigma</td>
			<td>E2375</td>
			<td>- used in combination with puro to assess general translation level 
			- used to assess SG connection with active translation 
			- dilute in water to 10 mg/mL</td>
		</tr>
		<tr>
			<td>Cycloheximide</td>
			<td>Sigma</td>
			<td>C4859</td>
			<td>- abbreviated CHX 
			- used to assess SG connection with active translation 
			- dilute in water to 10 mg/mL</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline</td>
			<td>Lonza / VWR</td>
			<td>95042-486</td>
			<td>- abbreviated PBS 
			- wash buffer for immunofluorescence</td>
		</tr>
		<tr>
			<td>Paraformaldehyde reagent grade, crystalline</td>
			<td>Sigma</td>
			<td>P6148-500G</td>
			<td>- make a 4% solution in hot PBS in fume hood, stir until dissolved 
			- aliquots can be stored at - 20 &#176;C for several months 
			- hazardous, use ventilation 
			- discard in special waste</td>
		</tr>
		<tr>
			<td>Methanol, ACS</td>
			<td>BDH / VWR</td>
			<td>BDH1135-4LP</td>
			<td>- pre-chilled to -20&#176;C before use 
			- discard in special waste</td>
		</tr>
		<tr>
			<td>Normal Horse Serum</td>
			<td>Thermo Fisher Scientific</td>
			<td>31874</td>
			<td>- abbreviated NHS 
			- dilute to 5% in PBS 
			- add sodium azide for storage at 4&#176;C 
			- blocking solution for immunofluorescence</td>
		</tr>
		<tr>
			<td>Ethanol, Pure, 200 Proof (100%),USP, KOPTEC</td>
			<td>Decon Labs / VWR</td>
			<td>89125-188</td>
			<td>- dilute to 70% with water</td>
		</tr>
		<tr>
			<td>Fisherbrand Superfrost Plus Microscope Slides</td>
			<td>Fisherbrand</td>
			<td>12-550-15</td>
			<td></td>
		</tr>
		<tr>
			<td>mouse anti G3BP1 antibody (TT-Y)</td>
			<td>Santa Cruz</td>
			<td>sc-81940</td>
			<td>- store at 4 &#176;C 
			- use at 1/100 dilution</td>
		</tr>
		<tr>
			<td>rabbit anti eIF4G antibody (H-300)</td>
			<td>Santa Cruz</td>
			<td>sc-11373</td>
			<td>- store at 4 &#176;C 
			- 1/250 dilution</td>
		</tr>
		<tr>
			<td>goat anti eIF3&#951; (N-20) antibody</td>
			<td>Santa Cruz</td>
			<td>sc-16377</td>
			<td>- eIF3&#951; is also known as eIF3b 
			- store at 4 &#176;C 
			- 1/250 dilution</td>
		</tr>
		<tr>
			<td>mouse anti puromycin 12D10 antibody</td>
			<td>Millipore</td>
			<td>MABE343</td>
			<td>- store at 4 &#176;C 
			- 1/1000 dilution</td>
		</tr>
		<tr>
			<td>Cy2 AffiniPure Donkey Anti-Mouse IgG (H+L)</td>
			<td>Jackson Immunoresearch</td>
			<td>715-225-150</td>
			<td>- reconstitute in water as per manufacturer&#8217;s instructions then store at 4&#176;C 
			- 1/250 dilution</td>
		</tr>
		<tr>
			<td>Cy3 AffiniPure Donkey Anti-Rabbit IgG (H+L)</td>
			<td>Jackson Immunoresearch</td>
			<td>711-165-152</td>
			<td>- reconstitute in water as per manufacturer&#8217;s instructions then store at 4&#176;C 
			- 1/2500 dilution</td>
		</tr>
		<tr>
			<td>Cy5 AffiniPure Donkey Anti-Goat IgG (H+L)</td>
			<td>Jackson Immunoresearch</td>
			<td>705-175-147</td>
			<td>- reconstitute in water as per manufacturer&#8217;s instructions then store at 4&#176;C 
			- 1/250 dilution</td>
		</tr>
		<tr>
			<td>Hoechst 33258 solution</td>
			<td>Sigma</td>
			<td>94403-1ML</td>
			<td>- incubate with secondary antibodies 
			-stock solution 0.5 mg/mL in dH20, protect from light 
			- Store at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Cy3 Streptavidin</td>
			<td>Jackson Immunoresearch</td>
			<td>016-160-084</td>
			<td>- reconstitute in water as per manufacturer&#8217;s instructions, then store at 4&#176;C 
			- 1/250 dilution</td>
		</tr>
		<tr>
			<td>oligo-(dT)40x probe biotinilated</td>
			<td>Integrated DNA Technologies (IDT)</td>
			<td>/</td>
			<td>- reconstitute in water to 100 ng/mL, aliquot and store at -20&#176;C 
			- dilute in hybridation buffer 1/50 prior to use 
			- custom order</td>
		</tr>
		<tr>
			<td>hybridation box</td>
			<td>/</td>
			<td>/</td>
			<td>- make a moisture chamber with any storing slide box by adding humidified paper on the bottom 
			- prewarm the box prior use</td>
		</tr>
		<tr>
			<td>20 x SSC buffer</td>
			<td>Thermo fisher Ambion</td>
			<td>AM9763</td>
			<td>- dilute to 2X SSC using RNase Free water (DEPC treated) 
			- store at room temperature 
			- wash buffer for FISH</td>
		</tr>
		<tr>
			<td>PerfectHybPlus Hybridization Buffer</td>
			<td>Sigma</td>
			<td>H7033-50ml</td>
			<td>- block and probe incubation buffer for FISH</td>
		</tr>
		<tr>
			<td>slide mounting media</td>
			<td>home made</td>
			<td>/</td>
			<td>- mix 5 g of &#8220;cold-soluble&#8221; poly(vinyl alcohol) in 20 ml of PBS 
			- mix by sonication, followed by stirring overnight at RT 
			- Add 5 ml of glycerol and 0.2 ml of 20 % sodium azide, and stir for 16 h at RT 
			- centrifugation at 20,000 g for 20 min, discard large pellet 
			- aliquot viscous liquid, long-term storage at -20&#176;C, 1 week at 4&#176;C</td>
		</tr>
		<tr>
			<td>Parafilm &#34;M&#34;</td>
			<td>Sigma</td>
			<td>P7793-1EA</td>
			<td>- usually referred as &#34;parafilm&#34;</td>
		</tr>
		<tr>
			<td>Poly(vinyl alcohol)</td>
			<td>Sigma</td>
			<td>P-8136-250G</td>
			<td>- reagent to make vinol</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma</td>
			<td>G5516-100ML</td>
			<td>- reagent to make vinol</td>
		</tr>
		<tr>
			<td>sodium azide</td>
			<td>Fisher Scientific</td>
			<td>S2002-5G</td>
			<td>- preservative agent for blocking solution and vinol 
			- make a 20 % dilution in water</td>
		</tr>
	</tbody>
</table>

methods to classify cytoplasmic foci as mammalian stress granules

细胞经常受到突然环境变化的挑战。应激颗粒（SGs​​），在暴露于应激条件的细胞中形成的细胞质核糖核蛋白复合物涉及细胞代谢和存活的各个方面。 SGs调节细胞信号通路，转录后基因表达和应激反应程序。这些含mRNA的颗粒的形成与细胞翻译直接相关。 SG组件由抑制翻译起始触发，SG拆卸通过翻译激活或抑制翻译延伸来促进。 SG组合进一步突出了这种关系。核心SG组分是失速的翻译起始前复合物，mRNA和选定的RNA结合蛋白（RBP）。 SG装配的目的是通过隔离翻译停滞的内部管理mRNA来保存细胞能，从而允许应激反应蛋白的增强翻译。另外对核心成分，如停滞的翻译早产复合物，SG含有大量的其他蛋白质和信号分子。 SG形成的缺陷可以削弱细胞对应激的适应性，从而促进细胞死亡。 SG和类似的含RNA颗粒已经与许多人类疾病相关，包括神经变性疾病和癌症，导致最近对分类和定义RNA颗粒亚型的兴趣。该方案描述了表征和量化哺乳动物SG的测定法。

应激诱发的灶不一定是SG。 SG被分类为含有mRNA，翻译起始因子和RNA结合蛋白的细胞质病灶，并且与主动翻译处于平衡状态。上述方案可用作模板来表征给定的应激是否诱导真正的 SG。 SG与其他已知的SG标记物共定位，包括蛋白质和mRNA两者。 SA和VRB诱导含有G3BP1，eIF4G和eIF3b的细胞质病灶（ 图2A ）。使用线扫?...

Watch this Scientific Journal Video about Methods to Classify Cytoplasmic Foci as Mammalian Stress Granules at JoVE.com

Methods to Classify Cytoplasmic Foci as Mammalian Stress Granules

应激颗粒（SG）是在暴露于各种应激的细胞中形成的非膜细胞质结构。 SGs包含mRNA，RNA结合蛋白，小核糖体亚基，翻译相关因子和各种细胞信号传导蛋白。该协议描述了一种使用几种实验方法来检测，表征和量化真正的 SG的工作流程。

将细胞质Foci分类为哺乳动物应激颗粒的方法

Cells are often challenged by sudden environmental changes. Stress Granules (SGs), cytoplasmic ribonucleoprotein complexes that form in cells exposed to ...

Research

JoVE Journal

Biology

Trypsinizing and Subculturing Mammalian Cells

Trypsinizing和传代的哺乳动物细胞

As cells reach confluency, they must be subcultured or passaged. Failure to subculture confluent cells results in reduced mitotic index and eventually in ...

科研

生物学

Quantification of &gamma;H2AX Foci in Response to Ionising Radiation

Quantification of ÃƒÅ½Ã‚Â³H2AX Foci in Response to Ionising Radiation

电离辐射的反应定量的γH2AX灶

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA ...

Quantitation of &gamma;H2AX Foci in Tissue Samples

Quantitation of ÃƒÅ½Ã‚Â³H2AX Foci in Tissue Samples

定量组织样本中的γH2AX灶

DNA double-strand breaks (DSBs) are particularly lethal and genotoxic lesions, that can arise either by endogenous (physiological or pathological) ...

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

激活细胞凋亡的细胞色素的细胞质显微注射 C</em

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1.  Apoptosis can be triggered when cells encounter a ...

Visualizing Cytoplasmic Flow During Single-cell Wound Healing in Stentor coeruleus

Visualizing Cytoplasmic Flow During Single-cell Wound Healing in Stentor coeruleus

可视化细胞质流动过程中单细胞伤口愈合<em&gt;蓝斑宏声</em

Although wound-healing is often addressed at the level of whole tissues, in many cases individual cells are able to heal wounds within themselves, ...

Visualization of G3BP Stress Granules Dynamics in Live Primary Cells

中G3BP应力颗粒动力学在Live原代细胞可视化

SGs can be visualized in cells by immunostaining of specific protein components or polyA+ mRNAs. SGs are highly dynamic and the study of their assembly ...

Stimulation of Cytoplasmic DNA Sensing Pathways In Vitro and In Vivo

Stimulation of Cytoplasmic DNA Sensing Pathways In Vitro and In Vivo

细胞质DNA的检测途径的刺激<em&gt;在体外</em&gt;和<em&gt;体内</em

In order to efficiently stimulate an innate immune response, DNA must be of sufficient length and purity. We present a method where double stranded DNA ...

Methods to Assess Subcellular Compartments of Muscle in C. elegans

Methods to Assess Subcellular Compartments of Muscle in C. elegans

方法来评估肌肉的亚细胞中<em&gt;温度。线虫</em

Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state. The loss of muscle mass and function with disease and age ...

Comet Assay as an Indirect Measure of Systemic Oxidative Stress

彗星试验系统性氧化应激的间接测量

Higher eukaryotic organisms cannot live without oxygen; yet, paradoxically, oxygen can be harmful to them. The oxygen molecule is chemically relatively ...

Xenopus laevis Egg Extract Preparation and Live Imaging Methods for Visualizing Dynamic Cytoplasmic Organization

Xenopus laevis Egg Extract Preparation and Live Imaging Methods for Visualizing Dynamic Cytoplasmic Organization

非洲爪蟾 用于可视化动态细胞质组织的蛋提取物制备和实时成像方法

Traditionally used for bulk biochemical assays, Xenopus laevis egg extracts have emerged as a powerful imaging-based tool for studying cytoplasmic ...