Anyaporn Sawasdichai Nazefah阿卜杜勒哈米德感谢泰王国政府和马来西亚政府，分别为博士奖学金。这项工作是由威康信托项目荣获PSJ和KG授予。我们也感谢布里斯托尔大学欧胜生物成像设施。

一，准备进行分馏本节介绍了聚- L -赖氨酸涂层显微镜的盖玻片和细胞附着之前分馏的准备。如果需要的细胞可以瞬时转染与蛋白表达载体之前或之后附件。 A.制备聚- L -赖氨酸涂布盖玻片<ol><li>准备一个1mg/ml的聚- L -赖氨酸的蒸馏水溶液。 </li><li>清洁盖玻片大衣与聚- L -赖氨酸孵化摇椅平台上至少1小时的解决方案，在22 ° C。 </li><li>用无菌蒸馏水冲洗涂盖玻片两次，并按照与单一，用96％乙醇洗。 </li><li>空气干燥的滤纸上涂盖玻片和保存以供将来使用的干货柜（干燥后，他们可堆叠）。 </li></ol> B.附加细胞，以盖玻片非贴壁细胞（这里我们使用K562细胞） <ol><li>在6孔板与涂层表面朝上放置在一个良好的聚- L -赖氨酸的涂布盖玻片。 </li><li>种子K562细胞在7x10 5个细胞的密度细胞/孔贝科的修改老鹰培养基（DMEM），辅以10％小牛血清（FBS），和PS（青霉素100units/ml，链霉素100mg/ml ）。在瞬时转染对K562细胞的情况下，可以进行播种前的细胞电（媒体200μL1 × 10 7细胞在250V /975μF0.4厘米电试管）。 </li><li>在37 ° C在5％的CO 2孵育24小时的细胞。 </li><li>倒掉的中期和细胞两次用冰冷的磷酸盐缓冲液（PBS）。 </li><li>按照与亚细胞分馏协议。 </li></ol>贴壁细胞（这里我们使用COS - 7细胞） <ol><li>在6孔板放置在一个良好的非涂布盖玻片。 </li><li>在PBS清洗两次预热在37 ° C </li><li>细胞分离与胰蛋白酶的PBS（0.03％EDTA，0.25％胰蛋白酶）消化在37 ° C为2至5分钟。重要的是不要过度消化的细胞，并尽快停止消化，主要是个人的浮动细胞存在。 </li><li>停止播种在3X10 5个细胞的密度细胞/孔的DMEM在6孔板与10％FBS和PSG（青霉素100单位/ ml，链霉素100ug/ml和L -谷氨酰胺292ug/ml）为辅的消化，正常的盖玻片。贴壁细胞一般附加到正常的显微镜盖玻片，但是，如果需要的话，可用于聚- L -赖氨酸涂布盖玻片。 </li><li>在37 ° C和5％的CO 2孵育24小时的细胞。在瞬时转染COS - 7细胞的情况下可以使用Fugene 6（罗氏）如 ​​果需要的话，并留下进一步的24小时内恢复在37的细胞° C和5％的CO 2。 </li><li>倒掉的中期和冰冷的PBS洗细胞两次。 </li><li>按照与亚细胞分馏协议。 </li></ol> 二。亚细胞分馏 原位分数是根据原理图如图1所示，是一个方法根据前面描述的5修改描述以前7，并在下面的详细协议。建议去除盖玻片取出液体前，每个分馏步骤转移到一个新的6孔板的盖玻片。虽然只有四个盖玻片成像所需的，额外的盖玻片须出示全细胞提取物和免疫印迹，如果这是在并行执行核基质组分。 <ol><li>准备和过滤器骨架缓冲液（CSK）：10mm的管道，300mm的蔗糖，氯化钠100MM，MgCl 2的3MM，为1mM EGTA 。 CSK缓冲液应新鲜配制的一天，分馏。 </li><li>如果需要从一个盖玻片的细胞可用于准备杂交西部全细胞提取物轻轻直接放置到盖玻片200ul工商业污水附加费（1％SDS，EDTA的2MM，20MM的Tris - HCl pH值7.4）缓冲和孵化为1或2分钟在22 ° C全细胞提取物可以被删除，蛋白质沉淀，加入250ul 1M（NH 4）2 SO 4冰孵化，直到其他西方样本准备。 </li><li>其余盖玻片用1ml冰冷的PBS洗两次在22 ° C的倾斜6板两次或三次。 </li><li>取出盖玻片，占整个细胞转移到一个新的6孔板。按照与细胞固定和免疫荧光协议。 </li><li>轻轻地清除余下的井的PBS。 </li><li>轻轻放置200ul CSK缓冲中提取的细胞质和松散举行的核蛋白质+ 0.1％（V / V）TRITON X - 100直接到其余每个盖玻片和孵化冰为1分钟。 </li><li>取出细胞​​质和松散举行的核提取物和沉淀步骤2中所述。 </li><li>用1ml冰冷的PBS冲洗盖玻片两次在22 °倾斜6板彗星。 </li><li>取出盖玻片，紧紧举行的核材料，并按照与细胞固定和免疫荧光协议。 </li><li>轻轻地清除余下的井的PBS，轻轻地放在+ 0.5％（V / V）Triton X - 100的的直接到其余每个盖玻片200ul CSK的缓冲区，并在冰上孵育20分钟，然后提取紧紧举行的蛋白质。 </li><li>取出紧密举行的提取物和沉淀步骤2中所述。 </li><li>用1ml冰冷的PBS冲洗盖玻片两次在22 °倾斜6板彗星。 </li><li>取出盖玻片染色分数，并按照与细胞固定和免疫荧光协议。 </li><li>轻轻地清除余下的井的PBS然后将200ul CSK缓冲+ 100ug/ml DNA酶我到剩余的盖玻片和30分钟的孵育在37 ° C。 </li><li>取出染色质的提取物和沉淀步骤2中所述。 </li><li>两次在22 ° C的倾斜板与冰冷的PBS冲洗盖玻片。 </li><li>取出盖玻片，核基质分数，并按照与细胞固定和免疫荧光协议。 </li><li>如果所需的基质蛋白，可以被删除，从西部的额外矩阵印迹使用步骤2中所述200ul工商业污水附加费缓冲区盖玻片。 </li><li>在替补出场的4个顶部的离心，免疫印迹的样品应在13000转离心° C和重悬于40ul SDS上样缓冲液（62.5mM的Tris - HCl pH值6.8，2％SDS（W / V），50毫米数码地面电视的颗粒， 10％甘油，0.01％溴酚蓝（W / V）），然后通过SDS - PAGE分析。 <img src="/files/ftp_upload/1958/1958fig1.jpg" alt="图1" /> 图1。一个示意图，在原地的亚细胞分馏。骨架缓冲液（CSK）由10mm的管道，300mm的蔗糖， 氯化钠 100MM，MgCl 2的3MM，为1mM EGTA。工商业污水附加费的缓冲区由1％SDS，2MM 20MM的Tris - HCl pH 7.4的EDTA，。 </li></ol>三。细胞固定和免疫荧光<ol><li>轻轻地添加4％的甲醛/ PBS 1ml到每个盖玻片，并在4 ° C孵育30分钟。 </li><li>每个盖玻片洗净1毫升的PBS轻轻的2-3倍在22 ° C </li><li>添加0.1％，海卫一X-100/PBS 400ul每个盖玻片，在22 ° C孵育10分钟。 </li><li>每个盖玻片洗净1毫升的PBS轻轻的2-3倍在22 ° C </li><li> 20分钟加3％的牛血清白蛋白（BSA）/ PBS和孵化400ul在22 ° C，以减少潜在的背景染色。 </li><li>每个盖玻片洗净1毫升的PBS轻轻的2-3倍在22 ° C </li><li>在PBS的主要抗体100ul直接到盖玻片，在22 ° C孵育1小时。小学和中学的抗体，应在PBS稀释至所需浓度。这可能需要为每个使用的抗体进行了优化。 </li><li>每个盖玻片洗净1毫升的PBS轻轻的2-3倍在22 ° C </li><li>将200ul的PBS孵育二级抗体在22 ° C间距离为1小时。 </li><li>每个盖玻片洗净1毫升的PBS轻轻的2-3倍在22 ° C </li><li>装载到一个玻璃显微镜幻灯片使用Vectashield安装培养基中含有的DAPI（4&#39;,6 - diamidino - 2 -苯基吲哚盐酸盐）每个盖玻片（细胞下跌）。 </li><li>从周围的盖玻片，用纸巾擦去多余的安装介质，并在22 ° C孵育至少30分钟，避光的幻灯片。 </li><li>修复盖玻片载玻片上，用丙烯酸指甲油。 </li><li>保持幻灯片避光前荧光显微镜可视化。 </li></ol>四。代表性的成果<img src="/files/ftp_upload/1958/1958fig2.jpg" alt="图2" /> 图2。非转染COS - 7细胞，亚细胞分馏，分馏的COS - 7细胞用甲醛固定，免疫染色使用的A / C抗体结合，以TRITC安装介质中含有的DAPI治疗α-微管蛋白，α-组蛋白H1或α-拉明。图片获得10倍的目镜和使用徕卡共聚焦显微镜63X物镜。细胞核使用DAPI荧光标记抗体染色的细胞在同一领域的可视化使用一个TRITC的过滤器设置的过滤器设置的可视化。 <img src="/files/ftp_upload/1958/1958fig3.jpg" alt="图3" /> 图3。一个GFP - 6E2的融合蛋白是与核基质 。COS - 7细胞表达绿色荧光蛋白（GFP）的融合，人类乳头状瘤病毒（HPV）6型组成的融合蛋白的亚细胞分馏E2蛋白（GFP - 6E2）。通过DAPI染色观察细胞核，而在转染细胞的GFP - 6E2本地化是直接通过GFP荧光的可视化。拉明一个/可视化使用α-核纤层蛋白A / C抗体结合TRITC和TRITC的过滤器设置。合并的图像显示，一些GFP - 6E2蛋白与核基质（右下面板）。在图2所描述的图像。

<ol>
	<li>Donaldson, M. M., Boner, W., Morgan, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TopBP1+regulates+human+papillomavirus+type+16+E2+interaction+with+chromatin.">TopBP1 regulates human papillomavirus type 16 E2 interaction with chromatin.</a> J Virol. 81, 4338-4342 (2007).</li><li>Javed, A., Guo, B., Hiebert, S., Choi, J. Y., Green, J., Zhao, S. C., Osborne, M. A., Stifani, S., Stein, J. L., Lian, J. B., van Wijnen, A. J., Stein, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Groucho/TLE/R-esp+proteins+associate+with+the+nuclear+matrix+and+repress+RUNX+(CBF(alpha)/AML/PEBP2(alpha))+dependent+activation+of+tissue-specific+gene+transcription.">Groucho/TLE/R-esp proteins associate with the nuclear matrix and repress RUNX (CBF(alpha)/AML/PEBP2(alpha)) dependent activation of tissue-specific gene transcription.</a> J Cell Sci. 113, 2221-2231 (2000).</li><li>Kowalczyk, A. M., Roeder, G. E., Green, K., Stephens, D. J., Gaston, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+the+induction+or+inhibition+of+apoptosis+by+HPV+proteins.">Measuring the induction or inhibition of apoptosis by HPV proteins.</a> Methods Mol. Med. 119, 419-432 (2005).</li><li>McLarren, K. W., Theriault, F. M., Stifani, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Association+with+the+nuclear+matrix+and+interaction+with+Groucho+and+RUNX+proteins+regulate+the+transcription+repression+activity+of+the+basic+helix+loop+helix+factor+Hes1.">Association with the nuclear matrix and interaction with Groucho and RUNX proteins regulate the transcription repression activity of the basic helix loop helix factor Hes1.</a> J Biol. Chem. 276, 1578-1584 (2001).</li><li>McNeil, S., Guo, B., Stein, J. L., Lian, J. B., Bushmeyer, S., Seto, E., Atchison, M. L., Penman, S., van Wijnen, A. J., Stein, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+of+the+YY1+transcription+factor+to+the+nucleolus+and+the+nuclear+matrix+in+situ:+the+C-terminus+is+a+principal+determinant+for+nuclear+trafficking.">Targeting of the YY1 transcription factor to the nucleolus and the nuclear matrix in situ: the C-terminus is a principal determinant for nuclear trafficking.</a> J Cell Biochem. 68, 500-510 (1998).</li><li>Melan, M. A., Sluder, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Redistribution+and+differential+extraction+of+soluble+proteins+in+permeabilized+cultured+cells.+Implications+for+immunofluorescence+microscopy.">Redistribution and differential extraction of soluble proteins in permeabilized cultured cells. Implications for immunofluorescence microscopy.</a> J Cell Sci. 101, 731-743 (1992).</li><li>Zou, N., Lin, B. Y., Duan, F., Lee, K. Y., Jin, G., Guan, R., Yao, G., Lefkowitz, E. J., Broker, T. R., Chow, L. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+hinge+of+the+human+papillomavirus+type+11+E2+protein+contains+major+determinants+for+nuclear+localization+and+nuclear+matrix+association.">The hinge of the human papillomavirus type 11 E2 protein contains major determinants for nuclear localization and nuclear matrix association.</a> J. Virol. 74, 3761-3770 (2000).</li></ol>

共同的问题和建议： 在清洗过程中的全部或大部分细胞都将丢失。在洗涤步骤中的液体必须避免盖玻片的6孔​​板边缓慢加入。同样的液体，应仔细倾斜板中删除，并慢慢地移液多余的液体。使用聚- L -赖氨酸涂布盖玻片，可提高细胞粘附。 基因组DNA是没有完全消化。对于某些类型的细胞的DNA酶I消化步骤，可能需要进行扩展和/或DNA酶I浓度增加，以达到彻...

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>BSA</td>
<td>Chemical</td>
<td>Sigma</td>
<td>A9647-100G</td>
<td>&nbsp;</td>
<tr>
<td>DNase I</td>
<td>Chemical</td>
<td>Sigma</td>
<td>DN25-1G</td>
<td>&nbsp;</td>
<tr>
<td>poly-L-Lysine</td>
<td>Chemical</td>
<td>Sigma</td>
<td>P-8920</td>
<td>&nbsp;</td>
<tr>
<td>Trypsin</td>
<td>Chemical</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>EGTA</td>
<td>Chemical</td>
<td>Sigma</td>
<td>E4378-25G </td>
<td>&nbsp;</td>
<tr>
<td>Fomaldehyde</td>
<td>Chemical</td>
<td>BDH</td>
<td>284216N</td>
<td>&nbsp;</td>
<tr>
<td>PIPES</td>
<td>Chemical</td>
<td>Sigma</td>
<td>P-6757</td>
<td>&nbsp;</td>
<tr>
<td>Sucrose</td>
<td>Chemical</td>
<td>Fisher Scientific</td>
<td>S/8600/53</td>
<td>&nbsp;</td>
<tr>
<td>Triton X-100</td>
<td>Chemical</td>
<td>Sigma</td>
<td>T-6876</td>
<td>&nbsp;</td>
<tr>
<td>Mounting Medium with DAPI</td>
<td>Chemical</td>
<td>Vector Laboratories</td>
<td>H-1200</td>
<td>&nbsp;</td>
<tr>
<td>Fugene 6 Transfection Reagent</td>
<td>Chemical</td>
<td>Roche </td>
<td>11814443001</td>
<td>&nbsp;</td>
<tr>
<td>Histone H1</td>
<td>Antibodies</td>
<td>Santa Cruz</td>
<td>SC-8030</td>
<td>&nbsp;</td>
<tr>
<td>Lamin A/C</td>
<td>Antibodies</td>
<td>Santa Cruz</td>
<td>SC-20681</td>
<td>&nbsp;</td>
<tr>
<td>Tubulin-&alpha;</td>
<td>Antibodies</td>
<td>Santa Cruz</td>
<td>SC-32293</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

in situ subcellular fractionation of adherent and non-adherent mammalian cells

蛋白质的功能是紧密耦合的蛋白定位。虽然一些蛋白质被限制到特定的位置或亚细胞车厢，许多蛋白质是自由扩散，作为一个自由交流的的一个亚群，是与特定的亚细胞域或结构紧密相关的人口。允许的可视化，在原地的亚细胞分馏蛋白质的条块分割，还可以揭示蛋白质亚群，定位于特定的结构。例如，去除可溶性的细胞质蛋白和松散举行的核蛋白质可以揭示一些与染色质的转​​录因子的稳定协会。随后的DNA消化，可以揭示在某些情况下，协会与网络，统称为核脚手架或核基质蛋白和RNA的。 在这里，我们描述了在显微镜盖玻片壁和非贴壁的哺乳动物细胞原位分馏过程中所需的步骤。使用特异性抗体或荧光融合蛋白和荧光显微镜，可以达到蛋白质的可视化。抗体和/或行为，作为特定的车厢或结构的标记的荧光染料允许蛋白定位进行详细的映射。原位分馏也可以结合免疫印迹比较每个分数中蛋白质的数额。这个简单的生化方法可以揭示协会，否则仍未被发现。

Watch this Scientific Journal Video about In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells at JoVE.com

In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells

允许在原位显微镜盖玻片上的哺乳动物细胞的亚细胞分馏蛋白定位可视化。

原位贴壁和非贴壁的哺乳动物细胞的亚细胞分馏

Protein function is intimately coupled to protein localization. Although some proteins are restricted to a specific location or subcellular compartment, ...

in-situ-subcellular-fractionation-adherent-non-adherent-mammalian

Research

JoVE Journal

Biology

In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells

15.7K 次观看.  University of Bristol. 允许在原位显微镜盖玻片上的哺乳动物细胞的亚细胞分馏蛋白定位可视化。

视频: In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells

Trypsinizing and Subculturing Mammalian Cells

As cells reach confluency, they must be subcultured or passaged. Failure to subculture confluent cells results in reduced mitotic index and eventually in cell death. The first step in subculturing is to detach cells from the surface of the primary culture vessel by trypsinization or mechanical means. The resultant cell suspension is then subdivided, or reseeded, into fresh cultures. Secondary cultures are checked for growth and fed periodically, and may be subsequently subcultured to produce tertiary cultures. The time between passaging of cells varies with the cell line and depends on the growth rate.

As cells reach confluency, they must be subcultured or passaged. This video will demonstrate a procedure for subculturing both adherent and suspension 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

MISSION esiRNA for RNAi Screening in Mammalian Cells

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as small interfering RNAs (siRNAs). The short double-stranded RNAs originate from longer double stranded precursors by the activity of Dicer, a protein of the RNase III family of endonucleases. The resulting fragments are components of the RNA-induced silencing complex (RISC), directing it to the cognate target mRNA. RISC cleaves the target mRNA thereby reducing the expression of the encoded protein1,2,3. RNAi has become a powerful and widely used experimental method for loss of gene function studies in mammalian cells utilizing small interfering RNAs.
Currently two main methods are available for the production of small interfering RNAs. One method involves chemical synthesis, whereas an alternative method employs endonucleolytic cleavage of target specific long double-stranded RNAs by RNase III in vitro. Thereby, a diverse pool of siRNA-like oligonucleotides is produced which is also known as endoribonuclease-prepared siRNA or esiRNA. A comparison of efficacy of chemically derived siRNAs and esiRNAs shows that both triggers are potent in target-gene silencing. Differences can, however, be seen when comparing specificity. Many single chemically synthesized siRNAs produce prominent off-target effects, whereas the complex mixture inherent in esiRNAs leads to a more specific knockdown10.
In this study, we present the design of genome-scale MISSION esiRNA libraries and its utilization for RNAi screening exemplified by a DNA-content screen for the identification of genes involved in cell cycle progression. We show how to optimize the transfection protocol and the assay for screening in high throughput. We also demonstrate how large data-sets can be evaluated statistically and present methods to validate primary hits. Finally, we give potential starting points for further functional characterizations of validated hits.

Here we use a human esiRNA library in a high-throughput screen for genes involved in cell division. We demonstrate how to set up and conduct an esiRNA screens, as well as how to analyze and validate the results.

在哺乳动物细胞的RNAi筛选使命esiRNA

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as ...

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

异源表达在哺乳动物细胞离子通道的突变和功能分析

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

Clonogenic Assay: Adherent Cells

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. Apart from documenting the method, the initial landmark study generated the first radiation-dose response curve for X-ray irradiated mammalian (HeLa) cells in culture1. Basically, the clonogenic assay enables an assessment of the differences in reproductive viability (capacity of cells to produce progeny; i.e. a single cell to form a colony of 50 or more cells) between control untreated cells and cells that have undergone various treatments such as exposure to ionising radiation, various chemical compounds (e.g. cytotoxic agents) or in other cases genetic manipulation. The assay has become the most widely accepted technique in radiation biology and has been widely used for evaluating the radiation sensitivity of different cell lines. Further, the clonogenic assay is commonly used for monitoring the efficacy of radiation modifying compounds and for determining the effects of cytotoxic agents and other anti-cancer therapeutics on colony forming ability, in different cell lines. A typical clonogenic survival experiment using adherent cells lines involves three distinct components, 1) treatment of the cell monolayer in tissue culture flasks, 2) preparation of single cell suspensions and plating an appropriate number of cells in petri dishes and 3) fixing and staining colonies following a relevant incubation period, which could range from 1-3 weeks, depending on the cell line. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cell lines with the use of an immortalized human keratinocyte cell line (FEP-1811)2. Also, our aims are to describe common features of clonogenic assays including calculation of the plating efficiency and survival fractions after exposure of cells to radiation, and to exemplify modification of radiation-response with the use of a natural antioxidant formulation.

The applicability of the clonogenic assay for evaluating reproductive viability has been established for more than 50 years. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cells.

克隆形成含量:贴壁细胞

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. ...

Analysis of Cell Cycle Position in Mammalian Cells

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of cell proliferation underlies the pathology of diseases like cancer. As such there is great need to be able to investigate cell proliferation and quantitate the proportion of cells in each phase of the cell cycle. It is also of vital importance to indistinguishably identify cells that are replicating their DNA within a larger population. Since a cell&prime;s decision to proliferate is made in the G1 phase immediately before initiating DNA synthesis and progressing through the rest of the cell cycle, detection of DNA synthesis at this stage allows for an unambiguous determination of the status of growth regulation in cell culture experiments.
DNA content in cells can be readily quantitated by flow cytometry of cells stained with propidium iodide, a fluorescent DNA intercalating dye. Similarly, active DNA synthesis can be quantitated by culturing cells in the presence of radioactive thymidine, harvesting the cells, and measuring the incorporation of radioactivity into an acid insoluble fraction. We have considerable expertise with cell cycle analysis and recommend a different approach. We Investigate cell proliferation using bromodeoxyuridine/fluorodeoxyuridine (abbreviated simply as BrdU) staining that detects the incorporation of these thymine analogs into recently synthesized DNA. Labeling and staining cells with BrdU, combined with total DNA staining by propidium iodide and analysis by flow cytometry1 offers the most accurate measure of cells in the various stages of the cell cycle. It is our preferred method because it combines the detection of active DNA synthesis, through antibody based staining of BrdU, with total DNA content from propidium iodide. This allows for the clear separation of cells in G1 from early S phase, or late S phase from G2/M. Furthermore, this approach can be utilized to investigate the effects of many different cell stimuli and pharmacologic agents on the regulation of progression through these different cell cycle phases.
In this report we describe methods for labeling and staining cultured cells, as well as their analysis by flow cytometry. We also include experimental examples of how this method can be used to measure the effects of growth inhibiting signals from cytokines such as TGF-&beta;1, and proliferative inhibitors such as the cyclin dependent kinase inhibitor, p27KIP1. We also include an alternate protocol that allows for the analysis of cell cycle position in a sub-population of cells within a larger culture5. In this case, we demonstrate how to detect a cell cycle arrest in cells transfected with the retinoblastoma gene even when greatly outnumbered by untransfected cells in the same culture. These examples illustrate the many ways that DNA staining and flow cytometry can be utilized and adapted to investigate fundamental questions of mammalian cell cycle control.

Determining the cell cycle position of a population of cells, or understanding how signals affect proliferation, can be readily measured by flow cytometry using this protocol. We report a simple experimental approach to staining cells and quantifying their position in the cell cycle.

在哺乳动物细胞中细胞周期位置分析

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of ...

Visualization of Endoplasmic Reticulum Localized mRNAs in Mammalian Cells

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum (ER). However, the visualization of these mRNAs can be challenging. This is especially true when only a fraction of the mRNA is ER-associated and their distribution to this organelle is obstructed by non-targeted (i.e. "free") transcripts. In order to monitor ER-associated mRNAs, we have developed a method in which cells are treated with a short exposure to a digitonin extraction solution that selectively permeabilizes the plasma membrane, and thus removes the cytoplasmic contents, while simultaneously maintaining the integrity of the ER. When this method is coupled with fluorescent in situ hybridization (FISH), one can clearly visualize ER-bound mRNAs by fluorescent microscopy. Using this protocol the degree of ER-association for either bulk poly(A) transcripts or specific mRNAs can be assessed and even quantified. In the process, one can use this assay to investigate the nature of mRNA-ER interactions.

Here we describe a method to visualize endoplasmic reticulum-associated mRNAs in mammalian tissue culture cells. This technique involves the selective permeabilization of the plasma membrane with digitonin to remove cytoplasmic contents followed by fluorescent in situ hybridization to detect either bulk poly(A) mRNA or specific transcripts.

在哺乳动物细胞中的内质网定位的mRNA的可视化

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum ...

Polysome Fractionation and Analysis of Mammalian Translatomes on a Genome-wide Scale

mRNA translation plays a central role in the regulation of gene expression and represents the most energy consuming process in mammalian cells. Accordingly, dysregulation of mRNA translation is considered to play a major role in a variety of pathological states including cancer. Ribosomes also host chaperones, which facilitate folding of nascent polypeptides, thereby modulating function and stability of newly synthesized polypeptides. In addition, emerging data indicate that ribosomes serve as a platform for a repertoire of signaling molecules, which are implicated in a variety of post-translational modifications of newly synthesized polypeptides as they emerge from the ribosome, and/or components of translational machinery. Herein, a well-established method of ribosome fractionation using sucrose density gradient centrifugation is described. In conjunction with the in-house developed &#8220;anota&#8221; algorithm this method allows direct determination of differential translation of individual mRNAs on a genome-wide scale. Moreover, this versatile protocol can be used for a variety of biochemical studies aiming to dissect the function of ribosome-associated protein complexes, including those that play a central role in folding and degradation of newly synthesized polypeptides.

Ribosomes play a central role in protein synthesis. Polyribosome (polysome) fractionation by sucrose density gradient centrifugation allows direct determination of translation efficiencies of individual mRNAs on a genome-wide scale. In addition, this method can be used for biochemical analysis of ribosome- and polysome-associated factors such as chaperones and signaling molecules.

多聚核糖体分离和哺乳动物Translatomes分析在基因组范围内

mRNA translation plays a central role in the regulation of gene expression and represents the most energy consuming process in mammalian cells. ...

Measurement of Heme Synthesis Levels in Mammalian Cells

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in various molecular and cellular processes such as gene transcription, translation, cell differentiation and cell proliferation. The biosynthesis levels of heme vary across different tissues and cell types and is altered in diseased conditions such as anemia, neuropathy and cancer. This technique uses [4-14C] 5-aminolevulinic acid ([14C] 5-ALA), one of the early precursors in the heme biosynthesis pathway to measure the levels of heme synthesis in mammalian cells. This assay involves incubation of cells with [14C] 5-ALA followed by extraction of heme and measurement of the radioactivity incorporated into heme. This procedure is accurate and quick. This method measures the relative levels of heme biosynthesis rather than the total heme content. To demonstrate the use of this technique the levels of heme biosynthesis were measured in several mammalian cell lines.

Altered intracellular heme levels are associated with common diseases such as cancer. Thus, there is a need to measure heme biosynthesis levels in diverse cells. The goal of this protocol is to provide a fast and sensitive method to measure and compare the levels of heme synthesis in different cells.

中血红素的合成水平在哺乳动物细胞中测量

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in ...

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, electrical excitability and cell proliferation. The diversity and specificity of Ca2+ signaling derives from mechanisms by which Ca2+ signals are generated to act over different time and spatial scales, ranging from cell-wide oscillations and waves occurring over the periods of minutes to local transient Ca2+ microdomains (Ca2+ puffs) lasting milliseconds. Recent advances in electron multiplied CCD (EMCCD) cameras now allow for imaging of local Ca2+ signals with a 128 x 128 pixel spatial resolution at rates of &#62;500 frames sec-1 (fps). This approach is highly parallel and enables the simultaneous monitoring of hundreds of channels or puff sites in a single experiment. However, the vast amounts of data generated (ca. 1 Gb per min) render visual identification and analysis of local Ca2+ events impracticable. Here we describe and demonstrate the procedures for the acquisition, detection, and analysis of local IP3-mediated Ca2+ signals in intact mammalian cells loaded with Ca2+ indicators using both wide-field epi-fluorescence (WF) and total internal reflection fluorescence (TIRF) microscopy. Furthermore, we describe an algorithm developed within the open-source software environment Python that automates the identification and analysis of these local Ca2+ signals. The algorithm localizes sites of Ca2+ release with sub-pixel resolution; allows user review of data; and outputs time sequences of fluorescence ratio signals together with amplitude and kinetic data in an Excel-compatible table.

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Here we present techniques for imaging local IP3-mediated Ca2+ events using fluorescence microscopy in intact mammalian cells loaded with Ca2+ indicators together with an algorithm that automates identification and analysis of these events.

成像本地CA<sup&gt; 2+</sup&gt;在培养的哺乳动物细胞信号

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

软件辅助跟踪动态细胞突变中蛋白质浓度的图形用户界面

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...