Name: cell cycle analysis in the c. elegans germline with the thymidine analog edu
Uploaded: 2018-10-22T14:00:00
Description: Watch this Scientific Journal Video about Cell Cycle Analysis in the C. elegans Germline with the Thymidine Analog EdU at JoVE.com

我们感谢 MG1693 的大肠杆菌库存中心;Wormbase;线虫遗传学中心, 由国立卫生研究院的研究基础设施项目 (P40OD010440) 资助, 用于菌株;统计咨询;陈爱萍丰试剂;Scharf, 安德烈. 施耐德, 桑迪普, 和约翰的培训, 建议, 支持和有益的讨论;Kornfeld 和施德实验室对这份手稿的反馈意见。这项工作由国家卫生研究院 (R01 AG02656106A1 到 KK、R01 GM100756 到 TS) 和国家科学基金会 predoctoral 奖学金 [DGE-1143954 和 DGE-1745038] 部分支持。无论是国家卫生研究院还是国家科学基金会, 在设计研究、收集、分析和解释数据方面, 都没有任何作用, 也没有写手稿。

1. 教育标记细菌的制备
<ol><li>
培养 MG1693 的初学者文化。大肠杆菌(大肠杆菌) MG1693 在 thyA 中携带突变。
	<ol>
<li>将大肠杆菌MG1693 从冷冻甘油储存到120毫米溶源性汤 (LB) 琼脂培养皿中。文化在37摄氏度过夜。</li>
		<li>从两个单独的大肠杆菌MG1693 菌落接种到两个重复的4毫升液体 LB 管, 37 摄氏度, 16 小时。 注意: MG1693 在 LB 中生长良好, 无需补充胸腺嘧啶或胸?...

<ol>
	<li>Fox, P. M., Vought, V. E., Hanazawa, M., Lee, M. -. H., Maine, E. M., Schedl, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclin+E+and+CDK-2+regulate+proliferative+cell+fate+and+cell+cycle+progression+in+the+C.+elegans+germline.">Cyclin E and CDK-2 regulate proliferative cell fate and cell cycle progression in the C. elegans germline.</a> Development. 138 (11), 2223-2234 (2011).</li><li>Crittenden, S. L., Leonhard, K. A., Byrd, D. T., Kimble, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+analyses+of+the+mitotic+region+in+the+Caenorhabditis+elegans+adult+germ+line.">Cellular analyses of the mitotic region in the Caenorhabditis elegans adult germ line.</a> Molecular biology of the cell. 17 (7), 3051-3061 (2006).</li><li>Seidel, H. S., Kimble, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-cycle+quiescence+maintains+Caenorhabditis+elegans+germline+stem+cells+independent+of+GLP-1/Notch.">Cell-cycle quiescence maintains Caenorhabditis elegans germline stem cells independent of GLP-1/Notch.</a> eLife. 4, (2015).</li><li>Fox, P. M., Schedl, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+Germline+Stem+Cell+Differentiation+Following+Loss+of+GLP-1+Notch+Activity+in+Caenorhabditis+elegans.">Analysis of Germline Stem Cell Differentiation Following Loss of GLP-1 Notch Activity in Caenorhabditis elegans.</a> Genetics. 201 (9), 167-184 (2015).</li><li>Kocsisova, Z., Kornfeld, K., Schedl, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+cycle+accumulation+of+the+proliferating+cell+nuclear+antigen+PCN-1+transitions+from+continuous+in+the+adult+germline+to+intermittent+in+the+early+embryo+of+C.+elegans.">Cell cycle accumulation of the proliferating cell nuclear antigen PCN-1 transitions from continuous in the adult germline to intermittent in the early embryo of C. elegans.</a> BMC Developmental Biology. 18 (1), (2018).</li><li>Salic, A., Mitchison, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+chemical+method+for+fast+and+sensitive+detection+of+DNA+synthesis+in+vivo.">A chemical method for fast and sensitive detection of DNA synthesis in vivo.</a> Proceedings of the National Academy of Sciences. 105 (7), 2415-2420 (2008).</li><li>vanden Heuvel, S., Kipreos, E. T. 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M., Engebrecht, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+timing+of+S+phases,+X+chromosome+replication,+and+meiotic+prophase+in+the+C.+elegans+germ+line.">Differential timing of S phases, X chromosome replication, and meiotic prophase in the C. elegans germ line.</a> Developmental Biology. 308 (1), 206-221 (2007).</li><li>Agarwal, I., Farnow, C., 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=HOP-1+presenilin+deficiency+causes+a+late-onset+notch+signaling+phenotype+that+affects+adult+germline+function+in+Caenorhabditis+elegans.">HOP-1 presenilin deficiency causes a late-onset notch signaling phenotype that affects adult germline function in Caenorhabditis elegans.</a> Genetics. 208 (2), 745-762 (2018).</li><li>Gumienny, T. L., Lambie, E., Hartwieg, E., Horvitz, H. 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J., Wei, Y., 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=Mitosis-specific+phosphorylation+of+histone+H3+initiates+primarily+within+pericentromeric+heterochromatin+during+G2+and+spreads+in+an+ordered+fashion+coincident+with+mitotic+chromosome+condensation.">Mitosis-specific phosphorylation of histone H3 initiates primarily within pericentromeric heterochromatin during G2 and spreads in an ordered fashion coincident with mitotic chromosome condensation.</a> Chromosoma. 106 (6), 348-360 (1997).</li><li>Zetka, M. C., Kawasaki, I., Strome, S., M&#252; Ller, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synapsis+and+chiasma+formation+in+Caenorhabditis+elegans+require+HIM-3,+a+meiotic+chromosome+core+component+that+functions+in+chromosome+segregation.">Synapsis and chiasma formation in Caenorhabditis elegans require HIM-3, a meiotic chromosome core component that functions in chromosome segregation.</a> Genes &amp; development. 13 (17), 2258-2270 (1999).</li><li>Hansen, D., Hubbard, E. J. A., Schedl, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi-pathway+control+of+the+proliferation+versus+meiotic+development+decision+in+the+Caenorhabditis+elegans+germline.">Multi-pathway control of the proliferation versus meiotic development decision in the Caenorhabditis elegans germline.</a> Developmental Biology. 268 (2), 342-357 (2004).</li><li>Crawley, O., Barroso, C., 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=Cohesin-interacting+protein+WAPL-1+regulates+meiotic+chromosome+structure+and+cohesion+by+antagonizing+specific+cohesin+complexes.">Cohesin-interacting protein WAPL-1 regulates meiotic chromosome structure and cohesion by antagonizing specific cohesin complexes.</a> eLife. 5 (2), 1-26 (2016).</li><li>Zhao, H., Halicka, H. D., 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=DNA+damage+signaling,+impairment+of+cell+cycle+progression,+and+apoptosis+triggered+by+5-ethynyl-2&#8242;-deoxyuridine+incorporated+into+DNA.">DNA damage signaling, impairment of cell cycle progression, and apoptosis triggered by 5-ethynyl-2&#8242;-deoxyuridine incorporated into DNA.</a> Cytometry Part A. 83 (11), 979-988 (2013).</li><li>Stiernagle, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maintenance+of+C.+elegans.">Maintenance of C. elegans.</a> WormBook. , 1-11 (2006).</li><li>Gervaise, A. L., Arur, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+and+Temporal+Analysis+of+Active+ERK.">Spatial and Temporal Analysis of Active ERK.</a> in the C. elegans Germline. Journal of Visualized Experiments. 117 (117), e54901-e54901 (2016).</li><li>Vos, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> De Cell Counter Plugin. , (2015).</li><li>Schindelin, J., Arganda-Carreras, I., 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=Fiji:+an+open-source+platform+for+biological-image+analysis.">Fiji: an open-source platform for biological-image analysis.</a> Nature Methods. 9 (7), 676-682 (2012).</li><li>Rasband, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> ImageJ. , (2016).</li><li>Michaelson, D., Korta, D. Z., Capua, Y., Hubbard, E. J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insulin+signaling+promotes+germline+proliferation+in+C.+elegans.">Insulin signaling promotes germline proliferation in C. elegans.</a> Development. 137 (4), 671-680 (2010).</li><li>Qin, Z., Jane, E., Hubbard, A., Hubbard, E. J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-autonomous+DAF-16/FOXO+activity+antagonizes+age-related+loss+of+C.+elegans+germline+stem/progenitor+cells.">Non-autonomous DAF-16/FOXO activity antagonizes age-related loss of C. elegans germline stem/progenitor cells.</a> Nature communications. 6 (5), 7107 (2015).</li><li>Luo, S., Kleemann, G. A., Ashraf, J. M., Shaw, W. M., Murphy, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TGFB+and+Insulin+Signaling+Regulate+Reproductive+Aging+via+Oocyte+and+Germline+Quality+Maintenance.">TGFB and Insulin Signaling Regulate Reproductive Aging via Oocyte and Germline Quality Maintenance.</a> Cell. 143 (2), 299-312 (2010).</li><li>Narbonne, P., Maddox, P. S., Labbe, J. -. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=daf-18/PTEN+locally+antagonizes+insulin+signalling+to+couple+germline+stem+cell+proliferation+to+oocyte+needs+in+C.+elegans.">daf-18/PTEN locally antagonizes insulin signalling to couple germline stem cell proliferation to oocyte needs in C. elegans.</a> Development. , 4230-4241 (2015).</li><li>Cinquin, A., Chiang, M., 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=Intermittent+Stem+Cell+Cycling+Balances+Self-Renewal+and.">Intermittent Stem Cell Cycling Balances Self-Renewal and.</a> Senescence of the C. elegans Germ Line. PLoS Genetics. 12 (4), 1005985 (2016).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Invitrogen EdU (5-ethynyl-2’-deoxyuridine). , 1-7 (2010).</li><li>Tuttle, A. H., Rankin, M. M., 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=Immunofluorescent+Detection+of+Two+Thymidine+Analogues+(CldU+and+IdU)+in+Primary+Tissue.">Immunofluorescent Detection of Two Thymidine Analogues (CldU and IdU) in Primary Tissue.</a> Journal of Visualized Experiments. 46 (46), e2166-e2166 (2010).</li></ol>

教育标记细菌的制备 (步骤 1) 对此协议至关重要, 也是解决问题的第一步。野生型年轻成人雌雄同体标签非常可靠地在一个4小时的教育脉冲, 使这对每一批新的教育标记细菌是一个有用的控制。此外, 完整的教育标记细菌进入肠道 (在老动物或某些咽/磨床缺陷突变体) 将标签与点击化学和显示为明亮的长方形斑点在肠道。用于标记雌雄同体的另一种技术是在3的高浓度 (1 mM) 中使用 ...

在动物发展中, 需要数以百计、数以千计、数以亿计的细胞分裂来形成成年有机体。细胞周期, 由 G1 (间隙)、S (合成)、G2 (间隙) 和 M (有丝分裂) 组成的细胞事件集定义了执行每个单元格除法的一系列事件。细胞周期是动态的, 最好是实时地得到赞赏, 这可能是技术上的困难。本协议中提供的技术允许一个人对静止图像进行细胞周期的相位和时间测量。
用核苷类似物 (如 5-乙炔-2 '-脱氧尿苷) 或 5-溴-2 '-脱氧尿苷 (BrdU) 进行标记, 是确定线虫线虫(线虫) 成人细胞周期动力学研究中 S 相的金标准。双性种系1,2,3,4,5。教育和 BrdU 几乎可以用于任何遗传背景, 因为它们不依赖任何基因结构。可视化 BrdU 需要苛刻的化学处理, 以暴露抗原的抗 BrdU 抗体染色, 这往往是不相容的评估其他细胞标记与其他抗体共同染色可视化。相比之下, 在轻度条件下, 通过单击化学进行可视化, 从而与抗体共染色6、7兼容。
标签的特异性是明确的, 因为原子核只在 S 阶段将胸苷 (5-乙炔-2 '-脱氧尿苷) 类似物纳入 DNA。可视化发生在固定组织中。该教育标签本身是无形的, 直到含有叠氮化物的染料或荧光基团与炔烃的铜催化单击化学8反应共价键。教育标签可以提供即时信息, 其中原子核是在 S 相, 使用短脉冲标记。教育还可以提供动态信息, 使用脉冲追逐或连续标记;例如, 在脉冲追逐实验中, 标签被稀释在每个细胞分裂或传播作为非分裂细胞进展通过开发。
线虫种系是一种强大的模型系统, 用于研究信号通路、干细胞、减数分裂和细胞周期。成人种系是一种极化装配线, 在远端发现了干细胞, 随后通过减数分裂前期进入和级数, 与配子的分期相协调 (图 1)。在近端, 卵母细胞成熟, 是排卵和受精和开始胚胎发生在子宫9,10,11。远端尖端细胞附近的约20个细胞直径长的区域, 包括有丝分裂循环种系茎、祖细胞和减数分裂的 S 相细胞, 而不是减数分裂前期的细胞, 被称为祖区2,4,9,12. 细胞膜在远端种系核之间不完全分离, 但祖细胞在很大程度上独立进行有丝分裂细胞循环。年轻成人雌雄同体种系祖区细胞中有丝分裂细胞周期的中位数为 6.5 h;G1 期短或不存在, 平静未观察到1、2、13。种系干细胞分化发生的主要原因是直接分化, 因此缺乏4的过境放大。在粗线期阶段的分化过程中, 大约4的5核不会形成卵母细胞, 而是通过细胞凋亡, 通过将胞质含量捐献给发育中的卵子12、14 , 作为护士体细胞。,15。
除了用核苷类似物标记 S 相的细胞外, 还可以使用抗体染色识别有丝分裂和减数分裂中的细胞。有丝分裂中的细胞核是免疫的抗磷组蛋白 H3 (Ser10) 抗体 (称为 pH3)7,16。减数分裂中的细胞核是免疫 anti-HIM-3 抗体 (减数分裂染色体轴蛋白)17。祖区的细胞核可以通过缺乏 HIM-3、nucleoplasmic REC-818的存在或 WAPL-119的存在来识别。WAPL-1 强度在体细胞性腺中最高, 祖区高, 早期减数分裂 prophases19。几个细胞周期测量是可能的, 在协议中的一些变化: I) 在 s 相和测量 s 相指数中识别核;II) 确定 m 相核, 测量 m 相指数;III) 确定核是否在有丝分裂或减数分裂的 S 阶段;IV) 测量 G2 的持续时间;V) 测量 G2+M+G1 阶段的 duartion;VI) 测量减数分裂的进入率;VII) 估计减数分裂的进展率。
一个可以做多细胞周期测量从几个类型的湿实验室实验。下面的协议描述了30分钟脉冲标记喂养C. 线虫成人雌雄同体与教育标记细菌和联合标记 M 相细胞通过染色与 anti-pH3 抗体和祖细胞的染色 anti-WAPL-1 抗体。只有在教育饲料的持续时间 (步骤 2.5), 使用的抗体类型 (步骤 5) 和分析 (步骤 8.3) 的变化在额外的测量需要。

<table border="0" cellpadding="0" cellspacing="0" style="width:1019px;" width="1020">
	<tbody>
		<tr height="42">
			<td >E. coli MG1693</td>
			<td style="width:255px;">Coli Genetic Stock Center</td>
			<td style="width:255px;">6411</td>
			<td style="width:255px;">grows fine in standard unsupplemented LB</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">E. coli OP50</td>
			<td style="width:255px;">Caenorhabditis Genetics Center</td>
			<td style="width:255px;">OP50</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Click-iT EdU Alexa Fluor 488 Imaging Kit</td>
			<td style="width:255px;">Thermo Fisher Scientific</td>
			<td style="width:255px;">C10337</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">5-Ethynyl-2&#8242;-deoxyuridine</td>
			<td style="width:255px;">Sigma</td>
			<td style="width:255px;">900584-50MG</td>
			<td style="width:255px;">or use EdU provided in kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Glucose</td>
			<td style="width:255px;">Sigma</td>
			<td style="width:255px;">D9434-500G</td>
			<td style="width:255px;">D-(+)-Dextrose</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Thiamine (Vitamin B1)</td>
			<td style="width:255px;">Sigma</td>
			<td style="width:255px;">T4625-5G</td>
			<td style="width:255px;">Reagent Grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Thymidine</td>
			<td style="width:255px;">Sigma</td>
			<td style="width:255px;">T1895-1G</td>
			<td style="width:255px;">BioReagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Magnesium sulfate heptahydrate</td>
			<td style="width:255px;">Sigma</td>
			<td style="width:255px;">M1880-1KG</td>
			<td style="width:255px;">MgSO4, Reagent Grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Sodium Phosphate, dibasic, anhydrous</td>
			<td style="width:255px;">Fisher</td>
			<td style="width:255px;">BP332-500G</td>
			<td style="width:255px;">Na2HPO4</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Potassium Phosphate, monobasic</td>
			<td style="width:255px;">Sigma</td>
			<td style="width:255px;">P5379-500G</td>
			<td style="width:255px;">KH2PO4</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Ammonium Chloride</td>
			<td style="width:255px;">Sigma</td>
			<td style="width:255px;">A4514-500G</td>
			<td style="width:255px;">NH4Cl, Reagent Plus</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Bacteriological Agar</td>
			<td style="width:255px;">US Biological</td>
			<td style="width:255px;">C13071058</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Calcium Chloride dihydrate</td>
			<td style="width:255px;">Sigma</td>
			<td style="width:255px;">C3881-500G</td>
			<td style="width:255px;">CaCl</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">LB Broth (Miller)</td>
			<td style="width:255px;">Sigma</td>
			<td style="width:255px;">L3522-1KG</td>
			<td style="width:255px;">Used at 25g/L</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Levamisole</td>
			<td style="width:255px;">Sigma</td>
			<td style="width:255px;">L9756-5G</td>
			<td style="width:255px;">0.241g/10ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Phosphate buffered saline</td>
			<td style="width:255px;">Calbiochem Omnipur</td>
			<td style="width:255px;">6506</td>
			<td style="width:255px;">homemade PBS works just as well</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Tween-20</td>
			<td style="width:255px;">Sigma</td>
			<td style="width:255px;">P1379-500ML</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">16% Paraformaldehyde, EM-grade ampules</td>
			<td style="width:255px;">Electron Microscopy Sciences</td>
			<td style="width:255px;">15710</td>
			<td style="width:255px;">10ml ampules</td>
		</tr>
		<tr height="62">
			<td height="62" style="height:62px;width:255px;">100% methanol</td>
			<td style="width:255px;">Thermo Fisher Scientific</td>
			<td style="width:255px;">A454-1L</td>
			<td style="width:255px;">Gold-label methanol is critical for proper morphology with certain antibodies</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Goat Serum</td>
			<td style="width:255px;">Gibco</td>
			<td style="width:255px;">16210-072</td>
			<td style="width:255px;">Lot 1671330</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">rabbit-anti-WAPL-1</td>
			<td style="width:255px;">Novus biologicals</td>
			<td style="width:255px;">49300002</td>
			<td style="width:255px;">Lot G3048-179A02, used at 1:2000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">mouse-anti-pH3 clone 3H10</td>
			<td style="width:255px;">Millipore</td>
			<td style="width:255px;">05-806</td>
			<td style="width:255px;">Lot#2680533, used at 1:500</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">goat-anti-rabbit IgG-conjugated Alexa Fluor 594</td>
			<td style="width:255px;">Invitrogen</td>
			<td style="width:255px;">A11012</td>
			<td style="width:255px;">Lot 1256147, used at 1:400</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">goat-anti-mouse IgG-conjugated Alexa Fluor 647</td>
			<td style="width:255px;">Invitrogen</td>
			<td style="width:255px;">A21236</td>
			<td style="width:255px;">Lot 1511347, used at 1:400</td>
		</tr>
		<tr height="62">
			<td height="62" style="height:62px;width:255px;">Vectashield antifade mounting medium containing 4&#39;,6-Diamidino-2-Phenylindole Dihydrochloride (DAPI)</td>
			<td style="width:255px;">Vector Laboratories</td>
			<td style="width:255px;">H-1200</td>
			<td style="width:255px;">mounting medium without DAPI can be used instead, following a separate DAPI incubation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">nail polish</td>
			<td style="width:255px;">Wet n Wild</td>
			<td style="width:255px;">DTC450B</td>
			<td style="width:255px;">any clear nail polish should work</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">S-medium</td>
			<td style="width:255px;">various</td>
			<td style="width:255px;"></td>
			<td style="width:255px;">see wormbook.org for protocol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">M9 buffer</td>
			<td style="width:255px;">various</td>
			<td style="width:255px;"></td>
			<td style="width:255px;">see wormbook.org for protocol</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">M9 agar</td>
			<td style="width:255px;">various</td>
			<td style="width:255px;"></td>
			<td style="width:255px;">same recipe as M9 buffer, but add 1.7% agar</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Nematode Growth Medium</td>
			<td style="width:255px;">various</td>
			<td style="width:255px;"></td>
			<td style="width:255px;">see wormbook.org for protocol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">dissecting watch glass</td>
			<td style="width:255px;">Carolina Biological</td>
			<td style="width:255px;">42300</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Parafilm laboratory film</td>
			<td style="width:255px;">Pechiney Plastic Packaging</td>
			<td style="width:255px;">PM-996</td>
			<td style="width:255px;">4 inch wide laboratory film</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">petri dishes</td>
			<td style="width:255px;"></td>
			<td style="width:255px;"></td>
			<td style="width:255px;">60 mm diameter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Long glass Pasteur pipettes</td>
			<td style="width:255px;"></td>
			<td style="width:255px;"></td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">1ml centrifuge tubes</td>
			<td style="width:255px;">MidSci Avant</td>
			<td style="width:255px;">2926</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Tips</td>
			<td style="width:255px;"></td>
			<td style="width:255px;"></td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Serological pipettes</td>
			<td style="width:255px;"></td>
			<td style="width:255px;"></td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">500 mL Erlenmyer flask</td>
			<td style="width:255px;"></td>
			<td style="width:255px;"></td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Aluminum foil</td>
			<td style="width:255px;"></td>
			<td style="width:255px;"></td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">25G 5/8&#8221; needles</td>
			<td style="width:255px;">BD PrecisionGlide&#160;</td>
			<td style="width:255px;">305122</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">5ml glass centrifuge tube</td>
			<td style="width:255px;">Pyrex&#160;</td>
			<td style="width:255px;"></td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Borosilicate glass tubes 1ml</td>
			<td style="width:255px;"></td>
			<td style="width:255px;"></td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">glass slides</td>
			<td style="width:255px;"></td>
			<td style="width:255px;"></td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">no 1 coverslips 22 x 40 mm</td>
			<td style="width:255px;"></td>
			<td style="width:255px;"></td>
			<td style="width:255px;">no 1.5 may work, also</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">37 &#176;C Shaker incubator</td>
			<td style="width:255px;"></td>
			<td style="width:255px;"></td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Tabletop Centrifuge</td>
			<td style="width:255px;"></td>
			<td style="width:255px;"></td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Clinical Centrifuge</td>
			<td style="width:255px;">IEC</td>
			<td style="width:255px;">428</td>
			<td style="width:255px;">with 6 swinging bucket rotor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Mini Centrifuge</td>
			<td style="width:255px;"></td>
			<td style="width:255px;"></td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:255px;">20 &#176;C incubator</td>
			<td style="width:255px;"></td>
			<td style="width:255px;"></td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:255px;">4 &#176;C refrigerator</td>
			<td style="width:255px;"></td>
			<td style="width:255px;"></td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">-20 &#176;C freezer</td>
			<td style="width:255px;"></td>
			<td style="width:255px;"></td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:255px;">Observer Z1 microscope</td>
			<td style="width:255px;">Zeiss</td>
			<td style="width:255px;"></td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Plan Apo 63X 1.4 oil-immersion objective lens</td>
			<td style="width:255px;">Zeiss</td>
			<td style="width:255px;"></td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:255px;">Ultraview Vox spinning disc confocal system</td>
			<td style="width:255px;">PerkinElmer&#160;</td>
			<td style="width:255px;"></td>
			<td style="width:255px;">Nikon spinning disc confocal system works very well, also, as described here: http://wucci.wustl.edu/Facilities/Light-Microscopy&#160;</td>
		</tr>
	</tbody>
</table>

cell cycle analysis in the c. elegans germline with the thymidine analog edu

真核细胞中的细胞周期分析经常利用染色体的形态、表达和/或定位所需的基因产品的各个阶段的细胞周期, 或合并核苷类似物。在 S 阶段, DNA 聚合酶将胸苷类似物 (如教育或 BrdU) 纳入染色体 DNA, 标记细胞进行分析。对于线虫, 核苷模拟教育在常规培养过程中被送入蠕虫, 并与免疫荧光技术兼容。种系是一种功能强大的模型系统, 用于研究信号通路、干细胞、减数分裂和细胞周期, 因为它是透明的、遗传的, 并且减数分裂前期和细胞分化/配子发生在线性装配样的时尚。这些特点使教育成为研究有丝分裂循环细胞和种系发展动态方面的一个很好的工具。该协议描述如何成功地准备教育细菌, 饲料他们到野生类型的线虫雌雄同体, 解剖双性性腺, 染色为教育纳入 DNA, 染色与抗体检测各种细胞周期和发展标记, 形象性腺和分析结果。该协议描述了测量 S 相指数、M 相指数、G2 持续时间、细胞周期持续时间、减数分裂率和减数分裂前期级数的方法和分析的变化。该方法可用于研究其他组织、阶段、遗传背景和生理条件下的细胞周期或细胞史。

由于 DNA 合成需要纳入教育, 人们可以得出结论, 在教育标记时间窗口中, 教育标记的核经历了 S 阶段。一个可以解释的细胞核, 标签在30分钟喂养与教育标记细菌作为细胞核在 S 阶段在解剖时。在较长的连续教育喂养实验中标签的细胞核在时间窗口和左 S 相, 或者可能已经标记在教育时间窗口的晚期。本地化信号与 DAPI 信号共用。在某些原子核中, 教育信号涵盖所有染色体, ?...

Watch this Scientific Journal Video about Cell Cycle Analysis in the C. elegans Germline with the Thymidine Analog EdU at JoVE.com

Cell Cycle Analysis in the C. elegans Germline with the Thymidine Analog EdU

本文描述了一种基于成像的方法, 可用于识别 S 相和分析种系中的细胞周期动力学. 此方法不需要转基因, 并且与免疫荧光染色兼容。

种系线虫与胸苷模拟教育的细胞周期分析

Cell cycle analysis in eukaryotes frequently utilizes chromosome morphology, expression and/or localization of gene products required for various phases ...

这种方法可以帮助回答有关C.elegans细胞周期和生殖系干细胞种群动力学的关键问题。该技术的主要优点是，它不需要转基因，它与免疫荧光染色兼容，它可以被修改为研究细胞在许多不同的条件下。当我第一次看到这种方法在Scheda实验室使用时，我非常兴奋。虽然这种方法可以提供对C.elegans细胞周期的洞察，它也可以应用于有关老化，营养，或改变基因型的更广泛问题。一般来说，新到这种方法的实验室可能与最初的白痴脱氧核糖核酸或C.elegans赤氧林染色斗争。首先使用无菌技术，在100毫升M9缓冲液中加入200微升10毫摩尔EdU，并适当补充4毫升新鲜生长的隔夜MG 1693大肠杆菌培养。使白痴需要EdU和胸腺素补充之间的微妙平衡。由于胸腺素的量必须足以使大肠杆菌长好，而EdU的量必须足以使坚固的屏蔽免受水平。在37摄氏度和200 RPM下不超过24小时后，使用无菌技术将培养培养在两到四个无菌的50毫升锥形管之间进行离心。重新涂抹四毫升新鲜 M9 缓冲液，使用相同的移液器将约 8 滴标有 E Coli MG1693 溶液的 E Coli MG1693 溶液涂抹到单个室温中心 60 毫升 M9 A 护式培养皿的中心。要将EdU标记的细菌喂给线虫，使用新鲜的PBS将线虫生长培养皿中的C.Elegans洗入1.5毫升的管子中，让动物通过重力短暂沉淀。用一毫升PBS清洗动物一到两次，用玻璃巴氏移液器将线虫转移到EdU草坪中心，用一小滴PBS。等待几分钟，让液体被吸收，并在20摄氏度下孵育至少30分钟。在孵化结束时，使用两毫升PBS将EdU培养盘上的蠕虫冲洗成玻璃解剖盘。解剖和固定线虫性腺后，冲洗一个小的毫升硅酸盐玻璃管和长玻璃巴氏移液器与PBS补充0.1%补间20，并使用移液器转移到管在少量的PBS补间。通过离心收集性腺，并使用长而拉出的玻璃牧场移液器去除尽可能多的超凡，而不会干扰性腺颗粒。对于 EdU 检测，在性腺中加入 100 微升点击 EdU 鸡尾酒，并在室温下用实验室膜覆盖管，进行 30 到 60 分钟的孵育。在孵育结束时，用100微升的反应冲洗缓冲液清洗性腺一次，用1毫升PBS补间洗涤4次。上次洗涤后，在样品中加入 25 微升防褪色安装介质，并辅以 DAPI。当介质在性腺上沉降时，将一个大的 2.5% 的 agarose 垫放在标准玻璃显微镜幻灯片上。然后用第二张幻灯片平展。接下来，使用长玻璃巴氏移液器，并将所有液体和性腺放在移液器的窄尖端，以尽量减少将性腺转移到垫片时样品损失。使用粘在牙签上的睫毛，将性腺分发到糖垫上，并去除任何灰尘颗粒。然后慢慢降低一个矩形玻璃盖滑到性腺上，注意避免气泡，并去除任何多余的溶液与实验室组织。在三维中可靠地计算细胞需要一些实践。使用斐济的单元格计数器插件和脚本（如标记单元格）删除任何重复项有助于使计数可重现。在允许盖滑在一夜之间结算后，使用细胞计数器插件和斐济手动计数每个原子核，根据磷石三个 EdU 或预基因区细胞标记的存在或不存在标记每个单独的原子核。EdU 信号与 DAPI 信号共存。在某些核中，EdU信号覆盖所有染色体，而在其他核中，EdU信号局部化为一到两个明亮的朋克，很可能在X染色体上，在S-相较晚时复制。EdU 信号从成功的 30 分钟标记定位到预基因区大约一半的核。虽然该技术在野生型年轻成年动物中一致有效，但相当一部分交配的5天大幼家未能在30分钟的EdU脉冲中标记。G2 的持续时间可以通过分析 M 相中 EdU 在时间过程中为正的核百分比来估计，从而允许计算 G2 的中位数和最大持续时间。G2 和 G1 的持续时间可以从 EdU 正的预基因区核的百分比进行估计，从而可以计算组合相的最大持续时间。在尝试此过程时，重要的是要记住使用同一批次的 EdU 菜，以最大限度地减少实验间变异性。按照这个程序，其他方法，如染色与额外的抗体可以执行或回答有关细胞周期，细胞命运，或信号通路的其他问题。不要忘记，使用杀核剂模拟和副甲酸抗剂可能是危险的，并且在执行此过程时应始终采取预防措施，例如戴硝基手套。

Research

JoVE Journal

Developmental Biology

Cell Cycle Analysis in the C. elegans Germline with the Thymidine Analog EdU

Comprehensive Assessment of Germline Chemical Toxicity Using the Nematode Caenorhabditis elegans

Comprehensive Assessment of Germline Chemical Toxicity Using the Nematode Caenorhabditis elegans

生殖系化学毒性的利用线虫综合评估<em&gt;秀丽隐杆线虫</em

Identifying the reproductive toxicity of the thousands of chemicals present in our environment has been one of the most tantalizing challenges in the ...

科研

发育生物学

Visualization of Chondrocyte Intercalation and Directional Proliferation via Zebrabow Clonal Cell Analysis in the Embryonic Meckel&#8217;s Cartilage

Visualization of Chondrocyte Intercalation and Directional Proliferation via Zebrabow Clonal Cell Analysis in the Embryonic MeckelÃƒÂ¢Ã¢â€šÂ¬Ã¢â€žÂ¢s Cartilage

通过在胚胎梅克尔软骨Zebrabow克隆细胞分析软骨插层和定向扩散的可视化

Development of the vertebrate craniofacial structures requires precise coordination of cell migration, proliferation, adhesion and differentiation. ...

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

斑马鱼幼虫骨骼肌诚信与伊文思蓝分析

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

Spatial and Temporal Analysis of Active ERK in the C. elegans Germline

Spatial and Temporal Analysis of Active ERK in the C. elegans Germline

在Active ERK的时空分析<em&gt;℃。线虫</em&gt;生殖系

The evolutionarily conserved&#160;extracellular signal transducing RTK-RAS-ERK pathway is an important kinase-signaling cascade that controls multiple ...

The C. elegans Intestine As a Model for Intercellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis at the Single-cell Level: Labeling by Antibody Staining, RNAi Loss-of-function Analysis and Imaging

The C. elegans Intestine As a Model for Intercellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis at the Single-cell Level: Labeling by Antibody Staining, RNAi Loss-of-function Analy

C. 线虫小肠作为细胞间腔形态发生和体内极化膜生物单细胞水平的模型: 抗体染色、rna 干扰功能分析和成像标记

Multicellular tubes, fundamental units of all internal organs, are composed of polarized epithelial or endothelial cells, with apical membranes lining the ...

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

rna 干扰和热休克诱导转录因子表达在重编程生殖细胞到神经元中的应用C. 线虫

Studying the cell biological processes during converting the identities of specific cell types provides important insights into mechanism that maintain ...

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

细胞聚集法评价果蝇切口与反式配体的结合及其对顺式配体的抑制作用

Notch signaling is an evolutionarily conserved cell-cell communication system used broadly in animal development and adult maintenance. Interaction of the ...

Three-dimensional Rendering and Analysis of Immunolabeled, Clarified Human Placental Villous Vascular Networks

Immunolabeled, 澄清人胎盘长柔毛血管网络的三维渲染与分析

Nutrient and gas exchange between mother and fetus occurs at the interface of the maternal intervillous blood and the vast villous capillary network that ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

秀丽线虫生殖的计算分析研究细胞核、蛋白质和细胞骨架的分布

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

Isolation and Staining of Mouse Skin Keratinocytes for Cell Cycle Specific Analysis of Cellular Protein Expression by Mass Cytometry

小鼠皮肤角质细胞的分离和染色,用于细胞周期特定分析细胞蛋白表达的量度细胞测定

The goal of this protocol is to detect and quantify protein expression changes in a cell cycle-dependent manner using single cells isolated from the ...