这项研究是由美国国立卫生研究院老龄研究所院内研究计划的支持。作者要感谢迈娅姆·戈罗斯佩和寇柏Abdelmohsen约衰老和寇柏Abdelmohsen的批判也读手稿许多有益的讨论。我们也感谢我们的实验室成员，特别是道格拉斯·德卢岑批判地阅读手稿。

作者什么都没有透露。

在这里，我们已经描述了使用人二倍体成纤维细胞用于复制和DNA损伤诱导的衰老的方法。此外，用于定量蛋白质和多种衰老相关蛋白的mRNA水平的技术包括，以及染色SA-β-gal和对DNA损伤标记物γ-H2AX。这些协议可广泛用于评估在体外和体内表型衰老，虽然用于体内 20表征衰老存在许多警告。其他细胞可表达衰老标记，即使它们不衰老细胞。例如，细胞如破骨细胞和巨噬细胞有较高水平的β-gal活性的32，33的。此外，许多癌细胞已经上调p16基因INK4A 34。细胞也可以积累的DNA损伤（ 例如，γ-H2AX焦点）和不衰老。 SASP因素也可以通过上调各种正常生理或可以或可以不涉及衰老细胞的病理状况。因此，多个衰老标记物应当利用在体内评估衰老时尤其如此。 虽然被发现衰老细胞在正常老化组织20余年前4增加，这是唯一的，现在我们充分理解角色的过多是衰老细胞在正常组织稳态和病理条件下发挥。最近的小鼠模型的就业，消除P16 INK4A阳性衰老细胞已经允许研究人员更清楚，更准确地领会衰老细胞的组织和器官35，36，37的作用。这些模型已经揭露了衰老细胞有助于与年龄有关的疾病，肿瘤发生的众多和鼠标寿命35 &lt;/ SUP&gt;，36，37。鉴于这些最新发现和衰老细胞的生理的重要性验证，时机已经成熟，在这些特定的细胞更多的研究。例如，研究用人策略，以治疗去除衰老细胞或延缓衰老可能是双方长期保健和寿命的研究是有益的。 最近，我们的实验室发现，二甲双胍，常用来治疗2型糖尿病的药物，减少细胞衰老38。如二甲双胍，目前正在提议作为一种抗衰老疗法，具有抗癌特性39，40，这将是未来的有趣的探索通过调控细胞衰老这种药物，或其他老化干预，是否会影响健康结果。 我们在这里讨论的协议是常见的，广泛的一ccepted技术来评估衰老表型。对于体外生长的细胞，关键是要监视由纺锤状至平坦的形貌细胞变化，以确保细胞是衰老（参见图1和2）。如果细胞不是阳性的SA-β-gal的或此处描述的其它标志物，在更高的细胞的PDL（对于复制衰老），或者在DNA损伤诱导的衰老之后增加的时间评估衰老。此外，这些协议可能需要为不同的细胞类型进行优化。用于评估SASP蛋白水平，至关重要的是，从细胞培养基中去除血清，因为这将导致在ELISA或蛋白质阵列高背景水平。因此，在设计实验时，记住这一点，因为它可能不是理想的同时评估多个标志。 染色SA-β-gal的，如在此所描述的，通过它需要细胞的固定的事实的限制。此外，细胞融合已经显示出影响SA-β-GAL活性。协议已经被开发使用的β-gal活性的荧光底物，其允许该标志物在使用流式细胞仪41，42，43活细胞的定量，以评估SA-β-Gal的活性。 SA-β-gal的活性也可以在使用化学发光方法44细胞裂解液中测定的。另外，γ-H2AX，也可以使用各种其他方法，包括流式细胞术或细胞裂解物45的免疫印迹测定。 应当指出的是，SAHF形成是细胞系相关的，并且主要是与相关联的癌基因诱导的衰老20,30，46。因此，SAHF可能不会出现在所有的衰老模型。的效用SAHF如在小鼠模型中衰老标记物也可通过其SAHF一般不小鼠衰老细胞30，46，47中观察到的事实的限制。在这里，针对的H3K9me2抗体被用于，但SAHF的其它分子标记可用于，包括的macroH2A，高迁移率族A（HMGA）蛋白，和三甲基化的赖氨酸9组蛋白H3（的H3K9me3）和结合的HP1蛋白。此外，染色质免疫沉淀测定法可以用于评估组蛋白标记或染色质结合蛋白与E2F靶基因的关联。 每个人都应该知道，有对衰老特征，包括异质性标志物，事实上，这些标记不衰老细胞中特异表达的各种限制。多个（在一般情况下，2 - 3）标记物应当用来评估衰老，包括这里未描述的标记。例如，DNA-疤痕，端粒损坏，和/或DNA损伤信号也可以定量。此外，如衰老和，特别地，所述SASP表型，最近已表明通过诱导衰老11，12，13中的特定应力源或损坏的影响，所以，以评估在每个实验范例多个标记是重要的。

半个多世纪前，海弗利克和他的同事描述了细胞是如何在未转化增殖的文化，但只是时间1有限的时期。人成纤维细胞的长期培养导致细胞增殖停止;然而，他们是代谢活跃，这被称为细胞衰老。衰老可用于抑制肿瘤发生是有利的，但它也可能是有害的，因为它被认为有助于其与衰老2,3发生的再生能力的损失。衰老细胞已被证明在组织中积累作为人类4年龄和在许多生物过程，包括胚胎发育，伤口愈合，组织修复，和年龄相关的炎症2的有牵连。 在培养细胞的持续传代引起复制性衰老，这已链接以端粒耗损和基因组不稳定性。各种细胞的应力，包括DNA损伤和癌基因，也可引起衰老3。由除端粒耗损等因素衰老经常被称为应力诱导或早衰和通常取决于P16 INK4A / Rb途径5。虽然增殖，未转化的细胞通常出现主轴在形状，衰老细胞可被识别为具有特定的特征，包括一平的，大的形态和提高衰老相关β半乳糖苷酶的活性（SA-β-GAL）（ 图1和2）。衰老细胞也积累的DNA损伤标记物，包括γ-H2AX（ 图3）6，以及潜在的衰老相关异灶（SAHF）（ 图4）7。衰老细胞中具有较高水平的细胞周期调节的，包含p<html> 16（P16 INK4A）和/或p21蛋白和p53（ 图5）8,9。此外，最近的数据已经表明，衰老细胞可通过分泌许多促炎性细胞因子和趋化因子具有非自主效应称为衰老相关分泌表型（SASP）10。虽然这种现象SASP可以从细胞类型而有所不同，以细胞类型，一般地，它是通过增加白细胞介素-6（IL-6），IL-8，粒细胞 - 巨噬细胞集落刺激因子（GM-CSF），生长 - 证明调节的原癌基因α（GRO-α），和GRO-β，等等（ 图6）。诱导衰老的特定应力或损坏还可能影响分泌表型11，12，13。 SASP可以通过测量使用的ELISA或细胞因子/蛋白质阵列分泌的蛋白质的水平来检测S = "外部参照"&gt; 10，14。尽管转录后的机制可以调节SASP蛋白水平11，15，16，17，在mRNA水平的变化也可以在许多情况下检测。这些变化通常更敏感，更容易比蛋白质水平的测量来量化。其它衰老标记物也可以被评估，包括持久性DNA损伤核灶，与染色质的改变称为DNA片段增强衰老（DNA-SCARS）18，以及各种其它的标记3，19，20。 在这里，我们将介绍常见的技术在培养细胞诱导的衰老，也为测量衰老的几个标志物，包括SA-β-Gal的，γ-H2AX，SAHF和衰老的蛋白和mRNANCE相关分子。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>16% Tris-glycine gels</td>
			<td>Invitrogen</td>
			<td colspan="2">XP00160BOX</td>
		</tr>
		<tr>
			<td>Acid-Phenol ChCl3</td>
			<td>Ambion</td>
			<td>AM9720</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa-Fluor 568 goat anti-mouse antibody</td>
			<td>Invitrogen</td>
			<td>A11031</td>
			<td>1:300 dilution</td>
		</tr>
		<tr>
			<td>Cell lifters</td>
			<td>Corning Inc.</td>
			<td>3008</td>
			<td>Cell scraper</td>
		</tr>
		<tr>
			<td>ECL anti-mouse HRP linked antibody</td>
			<td>Amersham</td>
			<td>NA931V</td>
			<td></td>
		</tr>
		<tr>
			<td>ECL Plus Western Blotting Substrate</td>
			<td>Pierce</td>
			<td>32132</td>
			<td>ECL</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Molecular Probes</td>
			<td>MP01306</td>
			<td>stock 5 mg/ml in dH2O</td>
		</tr>
		<tr>
			<td>GAPDH antibody</td>
			<td>Santa Cruz</td>
			<td>sc-32233</td>
			<td>1:1,000-5,000 dilution</td>
		</tr>
		<tr>
			<td>GlycoBlue</td>
			<td>Ambion</td>
			<td>AM9515</td>
			<td></td>
		</tr>
		<tr>
			<td>Histone H3 dimethyl K9 monoclonal antibody</td>
			<td>Abcam</td>
			<td>1220</td>
			<td>1:500 dilution</td>
		</tr>
		<tr>
			<td>Human IL-6 Quantikine ELISA assay</td>
			<td>R&#38;D systems</td>
			<td>D6050</td>
			<td></td>
		</tr>
		<tr>
			<td>Human IL-8 Quantikine ELISA assay</td>
			<td>R&#38;D systems</td>
			<td>D8000C</td>
			<td></td>
		</tr>
		<tr>
			<td>Human GROa Quantikine ELISA assay</td>
			<td>R&#38;D systems</td>
			<td>DRG00</td>
			<td></td>
		</tr>
		<tr>
			<td>N-N-dimethylformamide&#160;</td>
			<td>Sigma</td>
			<td>D4551</td>
			<td>DMF</td>
		</tr>
		<tr>
			<td>p16 monoclonal antibody</td>
			<td>BD Biosciences</td>
			<td>51-1325gr</td>
			<td>1:500 dilution</td>
		</tr>
		<tr>
			<td>p21 monoclonal antibody</td>
			<td>Millipore</td>
			<td>05-345</td>
			<td>1:750 dilution</td>
		</tr>
		<tr>
			<td>p53 monoclonal antibody</td>
			<td>Santa Cruz</td>
			<td>sc-126</td>
			<td>1:500 dilution clone DO-1</td>
		</tr>
		<tr>
			<td>phospho-H2AX (Ser139) FITC conjugate antibody</td>
			<td>Cell Signaling</td>
			<td>9719</td>
			<td>1:2000 dilution</td>
		</tr>
		<tr>
			<td>POWER SYBR-green PCR master mix&#160;</td>
			<td>Applied Biosystems</td>
			<td>4367659</td>
			<td></td>
		</tr>
		<tr>
			<td>Pre-stained molecular weight markers</td>
			<td>Biorad</td>
			<td>161-0374</td>
			<td></td>
		</tr>
		<tr>
			<td>ProLong Gold Antifade&#160;</td>
			<td>Invitrogen</td>
			<td>P36930</td>
			<td></td>
		</tr>
		<tr>
			<td>PVDF membrane&#160;</td>
			<td>Thermo Scientific</td>
			<td>88518</td>
			<td></td>
		</tr>
		<tr>
			<td>Senescence b-Galactosidase Staining Kit</td>
			<td>Cell Signaling</td>
			<td>9860</td>
			<td></td>
		</tr>
		<tr>
			<td>TRIzol</td>
			<td>Ambion/Life Tech</td>
			<td>10296028</td>
			<td></td>
		</tr>
	</tbody>
</table>

techniques to induce and quantify cellular senescence

响应于细胞应激或损害，增殖细胞可诱导特定的程序发起的长期细胞周期停滞的状态，称为细胞衰老。衰老细胞积累的有机体衰老和通过体外培养持续发生。衰老细胞中影响许多生物过程，包括胚胎发育，组织修复和再生，肿瘤抑制和老化。衰老细胞的标志包括，但不限于，增加的衰老相关β半乳糖苷酶的活性（SA-β-GAL）; P16 INK4A和p53和p21的水平;更高水平的DNA损伤，包括γ-H2AX;衰老相关异染色质灶（SAHF）的形成;和获取一个衰老相关分泌表型（SASP），这种现象的特征在于数促炎细胞因子和信号分子的分泌的。在这里，我们描述协议都复制和DNA损伤诱导的衰老中培养的细胞。另外，我们强调技术使用几种衰老相关的标志物，包括SA-β-GAL，γ-H2AX和SAHF染色监测衰老表型，并量化细胞周期调控和SASP因子蛋白和mRNA水平。这些方法可以适用于衰老的各种型号和组织的评估。

图2 -图6示出从SA-β-gal染色的代表性结果;染色γ-H2AX和SAHF; P16 INK4A，P21和p53的蛋白水平的评估;和mRNA和衰老相关分子的蛋白水平。增加SA-β-gal染色以复制和DNA损伤诱导的衰老发生。此外，观察与衰老发生形态变化。相比增殖的成纤维细胞的主轴外观细胞成为扩大且平坦。对于此实验范例，细胞染色和RNA /蛋白质被暴露于IR辐射之后，分离7天。更健壮的结果可以由IR暴露（ 即 10天）后等待再发生。条件需要为每个条件和试验设置确定。这些结果代表，并且当衰老使用其他方法诱导也可以被检测这些标记物。 图6中 ，基因名称列出并对应于CXCL1（GROα），CXCL2（GROβ），和CSF2（GM-CSF）。 IR诱导的衰老后观察到在WI-38细胞的一些mRNA水平的SASP因子的上调，但不是全部。其他衰老相关的mRNA，包括CDKN1A（编码p21蛋白）和CDKN2A（p16基因编码INK4A），被上调。其它mRNA（EDN1和ANKRD1）也被包括在内，先前已被证明在衰老细胞15要高。引物列于表1中 。 <img alt="图1" src="/files/ftp_upload/55533/55533fig1.jpg" /> 图1:增殖和衰老成纤维细胞。增殖（左）和衰老（右）的成纤维细胞的示意图。衰老诱导剂的实例示于叶llow框。衰老与上级指示标记的关联。 SA-β-GAL，衰老相关β半乳糖苷酶的活性; SASP，衰老相关分泌表型; SAHF，衰老相关异灶; DDR，DNA损伤应答。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55533/55533fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/55533/55533fig2.jpg" /> 图2.形态特征和衰老细胞的增加的SA-β-gal的活性的影响。 WI-38细胞，无论是增殖 "年轻"（PDL 27），复制衰老（PDL 40），或那些暴露于电离辐射（IR），进行染色SA-β-gal的活性。 IMR-90细胞中，无论是增殖 "年轻"（PDL 30），复制衰老（PDL 52），或那些暴露于电离辐射（IR），分别为STA独立非执行董事为SA-β-GAL活性。部分1 - 2描述诱导复制和DNA损伤诱导的衰老，和第3节说明了SA-β-gal染色。将细胞IR染色后7天。 CON，控制。比例尺=50μm以下。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55533/55533fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/55533/55533fig3.jpg" /> 图3.增加γ-H2AX焦点衰老细胞中。 WI-38细胞，无论是增殖 "年轻"（PDL 30）或复制衰老（PDL 53），固定，并用抗γ-H2AX的抗体和DAPI染色。注意γ-H2AX焦点的衰老细胞数量的增加。在共聚焦显微镜获得图像。 Z-切片截取，并使用该最大强度投影以获得一个单一的平面图像来可视化所有DETEC表灶。比例尺=20μm以下。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55533/55533fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/55533/55533fig4.jpg" /> 图衰老细胞SAHF 4.荧光染色。 （A）增殖WI-38细胞（PDL 27）不接受治疗，或暴露于电离辐射（IR）。 10天后，将细胞用DAPI染色以显现SAHF（注意在IR处理的细胞密/点状DAPI染色）（B）IDH4是在地塞米松存在下生长（增殖）或木炭处理的FBS诱导衰老（衰老）。 10天后，将细胞用针对的H3K9me2和DAPI的抗体染色。在共聚焦显微镜获得图像。 Z-切片截取，并使用最大强度投影以获得一个单一的平面图片。在（A）和（B）的增殖细胞具有漫DAPI染色，而衰老细胞具有点状DAPI染色。在（B）中，控制单元具有用于漫的H3K9me2，很轻染色，而染色的H3K9me2更坚固，并与在衰老细胞DAPI染色共定位病灶。比例尺=20μm以下。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55533/55533fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/55533/55533fig5.jpg" /> 图5.定量衰老相关蛋白。 （ - ）或暴露于电离辐射的10格雷（IR）IMR-90细胞中的蛋白质是从任一增殖"年轻"（PDL 30）IMR-90细胞中分离。将培养基换成每48小时，将细胞IR裂解后7天。样品通过SDS-PAGE分析，具有抗P16 INK4A antibo免疫印迹模具，并与抗p21再p53抗体再探测。 GAPDH用作蛋白质样对照。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55533/55533fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图6" src="/files/ftp_upload/55533/55533fig6.jpg" /> 图6.定量衰老相关的mRNA和蛋白质SASP的。 （ - ），其暴露于电离辐射（IR）的10戈瑞或WI-38细胞（A）RNA是从任一增殖"年轻"（PDL 27或34）WI-38细胞中分离。培养基每48个小时改变和IR后进行RNA分离7天。 RT-qPCR的用于定量使用表1中列出的基因特异性引物所指示的衰老相关的mRNA的水平。 18S水平用于标准化。直方图代表平均标准化ŧ从5个独立实验ö未处理±SEM。 （B）WI-38细胞（PDL 26）暴露于IR的10戈瑞; 10d中后，将培养基换成无血清培养基（参见第7节）。 24个小时后，收集条件培养基，并使用ELISA测定IL-6，IL-8，GROα和水平进行定量。直方图表示3个实验的平均值±SEM。 * P &lt;0.05和*** P &lt;0.001通过学生t检验。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55533/55533fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 人类引物 </td><td> 前锋 </td><td> 相反 </td></tr><tr><td> 18S </td><td> CCCTATCAACTTTCGATGGTAGTCG </td><td> CCAATGGATCCTCGTTAAAGGATTT </td></tr><tr><td> ANKRD1 </td><td> AGT AGA GGA ACT GGT CACTGG </td><td> TGG GCT AGA AGT GTCTTC AGAŧ </td></tr><tr><td> CDKN1A（P21） </td><td> GAC ACCACT GGA GGG TGA CT </td><td> CAGGTC CAC ATG GTC TTC CT </td></tr><tr><td> CDKN2A（P16） </td><td> CCAACGCACCGAATAGTTACG </td><td> GCGCTGCCCATCATCATG </td></tr><tr><td> CSF2（GM-CSF） </td><td> GGCCCCTTGACCATGATG </td><td> TCTGGGTTGCACAGGAAGTTT </td></tr><tr><td> CXCL1 </td><td> GAAAGCTTGCCTCAATCCTG </td><td> CACCAGTGAGCTTCCTCCTC </td></tr><tr><td> CXCL2 </td><td> AACTGCGCTGCCAGTGCT </td><td> CCCATTCTTGAGTGTGGCTA </td></tr><tr><td> EDN1 </td><td> CAG CAGTCT TAG GCG CTG AG </td><td> ACTCTT TAT CCA TCA GGG ACG AG </td></tr><tr><td> IL6 </td><td> CCGGGAACGAAAGAGAAGCT </td><td> GCGCTTGTGGAGAAGGAGTT </td></tr><tr><td> IL7 </td><td> CTCCAGTTGCGGTCATCATG </td><td> GAGGAAGTCCAAAGATATACCTAAAAGAA </tD&gt; </tr><tr><td> IL8 </td><td> CTTTCCACCCCAAATTTATCAAAG </td><td> CAGACAGAGCTCTCTTCCATCAGA </td></tr></tbody></table> 表1:RT-qPCR 的引物对衰老相关的mRNA。正向和反向引物使用RT-qPCR的SYBR绿色技术表示。

Watch this Scientific Journal Video about Techniques to Induce and Quantify Cellular Senescence at JoVE.com

Techniques to Induce and Quantify Cellular Senescence

细胞衰老，细胞周期停滞的不可逆状态，可通过各种细胞应激来诱导。在这里，我们描述的协议来诱导细胞衰老和方法来评估衰老的标志。

技术诱导和量化细胞衰老

In response to cellular stress or damage, proliferating cells can induce a specific program that initiates a state of long-term cell-cycle arrest, termed ...

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32.9K 次观看.  National Institute on Aging, National Institutes of Health. 细胞衰老，细胞周期停滞的不可逆状态，可通过各种细胞应激来诱导。在这里，我们描述的协议来诱导细胞衰老和方法来评估衰老的标志。 

视频: Techniques to Induce and Quantify Cellular Senescence

BioMEMS and Cellular Biology: Perspectives and Applications

The ability to culture cells has revolutionized hypothesis testing in basic cell and molecular biology research. It has become a standard methodology in drug screening, toxicology, and clinical assays, and is increasingly used in regenerative medicine. However, the traditional cell culture methodology   essentially consisting of the immersion of a large population of cells in a homogeneous fluid medium and on a homogeneous flat substrate   has become increasingly limiting both from a fundamental and practical perspective. Microfabrication technologies have enabled researchers to design, with micrometer control, the biochemical composition and topology of the substrate, and the medium composition, as well as the neighboring cell type in the surrounding cellular microenvironment. Additionally, microtechnology is conceptually well-suited for the development of fast, low-cost in vitro systems that allow for high-throughput culturing and analysis of cells under large numbers of conditions. In this interview, Albert Folch explains these limitations, how they can be overcome with soft lithography and microfluidics, and describes some relevant examples of research in his lab and future directions.

微机电与细胞生物学研究所：展望与应用

The ability to culture cells has revolutionized hypothesis testing in basic cell and molecular biology research. It has become a standard methodology in ...

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生物学

Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo

This protocol describes the use of the Drosophila melanogaster syncytial embryo for studying mitosis1. Drosophila has useful genetics with a sequenced genome, and it can be easily maintained and manipulated. Many mitotic mutants exist, and transgenic flies expressing functional fluorescently (e.g. GFP) - tagged mitotic proteins have been and are being generated. Targeted gene expression is possible using the GAL4/UAS system2. 

The Drosophila early embryo carries out multiple mitoses very rapidly (cell cycle duration, &asymp;10 min). It is well suited for imaging mitosis, because during cycles 10-13, nuclei divide rapidly and synchronously without intervening cytokinesis at the surface of the embryo in a single monolayer just underneath the cortex. These rapidly dividing nuclei probably use the same mitotic machinery as other cells, but they are optimized for speed; the checkpoint is generally believed to not be stringent, allowing the study of mitotic proteins whose absence would cause cell cycle arrest in cells with a strong checkpoint. Embryos expressing GFP labeled proteins or microinjected with fluorescently labeled proteins can be easily imaged to follow live dynamics (Fig. 1). In addition, embryos can be microinjected with function-blocking antibodies or inhibitors of specific proteins to study the effect of the loss or perturbation of their function3. These reagents can diffuse throughout the embryo, reaching many spindles to produce a gradient of concentration of inhibitor, which in turn results in a gradient of defects comparable to an allelic series of mutants. Ideally, if the target protein is fluorescently labeled, the gradient of inhibition can be directly visualized4. It is assumed that the strongest phenotype is comparable to the null phenotype, although it is hard to formally exclude the possibility that the antibodies may have dominant effects in rare instances, so rigorous controls and cautious interpretation must be applied. Further away from the injection site, protein function is only partially lost allowing other functions of the target protein to become evident.

Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo

This protocol describes the use of microinjection and high resolution imaging in the Drosophila melanogaster syncytial embryo to study mitosis.

研究有丝分裂的显微注射技术果蝇合胞体胚胎

This protocol describes the use of the Drosophila melanogaster syncytial embryo  for studying mitosis1.  Drosophila has useful genetics with a sequenced ...

Imaging Mismatch Repair and Cellular Responses to DNA Damage in Bacillus subtilis

Both prokaryotes and eukaryotes respond to DNA damage through a complex set of physiological changes. Alterations in gene expression, the redistribution of existing proteins, and the assembly of new protein complexes can be stimulated by a variety of DNA lesions and mismatched DNA base pairs. Fluorescence microscopy has been used as a powerful experimental tool for visualizing and quantifying these and other responses to DNA lesions and to monitor DNA replication status within the complex subcellular architecture of a living cell. Translational fusions between fluorescent reporter proteins and components of the DNA replication and repair machinery have been used to determine the cues that target DNA repair proteins to their cognate lesions in vivo and to understand how these proteins are organized within bacterial cells. In addition, transcriptional and translational fusions linked to DNA damage inducible promoters have revealed which cells within a population have activated genotoxic stress responses. In this review, we provide a detailed protocol for using fluorescence microscopy to image the assembly of DNA repair and DNA replication complexes in single bacterial cells. In particular, this work focuses on imaging mismatch repair proteins, homologous recombination, DNA replication and an SOS-inducible protein in Bacillus subtilis. All of the procedures described here are easily amenable for imaging protein complexes in a variety of bacterial species.

Imaging Mismatch Repair and Cellular Responses to DNA Damage in Bacillus subtilis

A detailed protocol is described for imaging the real time formation of DNA repair complexes in Bacillus subtilis cells.

成像错配修复和DNA损伤的细胞反应枯草芽孢杆菌</em

Both prokaryotes and eukaryotes respond to DNA damage through a complex set of physiological changes. Alterations in gene expression, the redistribution ...

Aseptic Laboratory Techniques: Plating Methods

Microorganisms are present on all inanimate surfaces creating ubiquitous sources of possible contamination in the laboratory. Experimental success relies on the ability of a scientist to sterilize work surfaces and equipment as well as prevent contact of sterile instruments and solutions with non-sterile surfaces. Here we present the steps for several plating methods routinely used in the laboratory to isolate, propagate, or enumerate microorganisms such as bacteria and phage. All five methods incorporate aseptic technique, or procedures that maintain the sterility of experimental materials. Procedures described include (1) streak-plating bacterial cultures to isolate single colonies, (2) pour-plating and (3) spread-plating to enumerate viable bacterial colonies, (4) soft agar overlays to isolate phage and enumerate plaques, and (5) replica-plating to transfer cells from one plate to another in an identical spatial pattern. These procedures can be performed at the laboratory bench, provided they involve non-pathogenic strains of microorganisms (Biosafety Level 1, BSL-1). If working with BSL-2 organisms, then these manipulations must take place in a biosafety cabinet. Consult the most current edition of the Biosafety in Microbiological and Biomedical Laboratories (BMBL) as well as Material Safety Data Sheets (MSDS) for Infectious Substances to determine the biohazard classification as well as the safety precautions and containment facilities required for the microorganism in question. Bacterial strains and phage stocks can be obtained from research investigators, companies, and collections maintained by particular organizations such as the American Type Culture Collection (ATCC). It is recommended that non-pathogenic strains be used when learning the various plating methods. By following the procedures described in this protocol, students should be able to: 
 
&#9679; Perform plating procedures without contaminating media. 
&#9679; Isolate single bacterial colonies by the streak-plating method. 
&#9679; Use pour-plating and spread-plating methods to determine the concentration of bacteria. 
&#9679; Perform soft agar overlays when working with phage. 
&#9679; Transfer bacterial cells from one plate to another using the replica-plating procedure. 
&#9679; Given an experimental task, select the appropriate plating method.

When working with media and reagents used to culture microorganisms, aseptic technique must be practiced to ensure contamination is minimized. A variety of plating methods are routinely used to isolate, propagate, or enumerate bacteria and phage, all of which incorporate procedures that maintain the sterility of experimental materials.

无菌实验室技术:电镀方法

Microorganisms are present on all inanimate surfaces creating ubiquitous sources of possible contamination in the laboratory. Experimental success relies ...

Telomere Length and Telomerase Activity; A Yin and Yang of Cell Senescence

Telomeres are repeating DNA sequences at the tip ends of the chromosomes that are diverse in length and in humans can reach a length of 15,000 base pairs. The telomere serves as a bioprotective mechanism of chromosome attrition at each cell division. At a certain length, telomeres become too short to allow replication, a process that may lead to chromosome instability or cell death. Telomere length is regulated by two opposing mechanisms: attrition and elongation. Attrition occurs as each cell divides. In contrast, elongation is partially modulated by the enzyme telomerase, which adds repeating sequences to the ends of the chromosomes. In this way, telomerase could possibly reverse an aging mechanism and rejuvenates cell viability. These are crucial elements in maintaining cell life and are used to assess cellular aging. In this manuscript we will describe an accurate, short, sophisticated and cheap method to assess telomere length in multiple tissues and species. This method takes advantage of two key elements, the tandem repeat of the telomere sequence and the sensitivity of the qRT-PCR to detect differential copy numbers of tested samples. In addition, we will describe a simple assay to assess telomerase activity as a complementary backbone test for telomere length.

An accurate, short, sophisticated and cheap method is described that assesses telomere length in multiple tissues and species using qRT-PCR. In addition, we will describe a simple assay to assess telomerase activity as a complementary backbone test for telomere length.

端粒长度和端粒酶活性;阴阳细胞衰老

Telomeres  are repeating DNA sequences at the tip ends of the chromosomes that are diverse  in length and in humans can reach a length of 15,000 base ...

Simple and Effective Administration and Visualization of Microparticles in the Circulatory System of Small Fishes Using Kidney Injection

The systemic administration of micro-size particles into a living organism can be applied for vasculature visualization, drug and vaccine delivery, implantation of transgenic cells and tiny optical sensors. However, intravenous microinjections into small animals, which are mostly used in biological and veterinary laboratories, are very difficult and require trained personnel. Herein, we demonstrate a robust and efficient method for the introduction of microparticles into the circulatory system of adult zebrafish (Danio rerio) by injection into the fish kidney. To visualize the introduced microparticles in the vasculature, we propose a simple intravital imaging technique in fish gills. In vivo monitoring of the zebrafish blood pH was accomplished using an injected microencapsulated fluorescent probe, SNARF-1, to demonstrate one of the possible applications of the described technique. This article provides a detailed description of the encapsulation of pH-sensitive dye and demonstrates the principles of the quick injection and visualization of the obtained microcapsules for in vivo recording of the fluorescent signal. The proposed method of injection is characterized by a low mortality rate (0-20%) and high efficiency (70-90% success), and it is easy to institute using commonly available equipment. All described procedures can be performed on other small fish species, such as guppies and medaka.

This article demonstrates the principles of a quick, minimally invasive injection of fluorescent microparticles into the circulatory system of small fishes and the in vivo visualization of the microparticles in fish blood.

小鱼肾注射液循环系统中微粒的简易有效管理与可视化

The systemic administration of micro-size particles into a living organism can be applied for vasculature visualization, drug and vaccine delivery, ...

Nasal Potential Difference to Quantify Trans-epithelial Ion Transport in Mice

The nasal potential difference test has been used for almost three decades to assist in the diagnosis of cystic fibrosis (CF). It has proven to be helpful in cases of attenuated, oligo- or mono-symptomatic forms of CF usually diagnosed later in life, and of CF-related disorders such as congenital bilateral absence of vas deferens, idiopathic chronic pancreatitis, allergic bronchopulmonary aspergillosis, and bronchiectasis. In both clinical and preclinical settings, the test has been used as a biomarker to quantify responses to targeted therapeutic strategies for CF. Adapting the test to a mouse is challenging and can entail an associated mortality. This paper describes the adequate depth of anesthesia required to maintain a nasal catheter in situ for continuous perfusion. It lists measures to avoid broncho-aspiration of solutions perfused in the nose. It also describes the animal care at the end of the test, including administration of a combination of antidotes of the anesthetic drugs, leading to rapidly reversing the anesthesia with full recovery of the animals. Representative data obtained from a CF and a wild-type mouse show that the test discriminates between CF and non-CF. Altogether, the protocol described here allows reliable measurements of the functional status of trans-epithelial chloride and sodium transporters in spontaneously breathing mice, as well as multiple tests in the same animal while reducing test-related mortality.

Here, we present a protocol to measure nasal potential difference in mice. The test quantifies the function of transmembrane ion transporters such as the cystic fibrosis transmembrane conductance regulator and the epithelial sodium channel. It is valuable to evaluate the efficacy of novel therapies for cystic fibrosis.

鼻电位差量化小鼠跨上皮离子转运

The nasal potential difference test has been used for almost three decades to assist in the diagnosis of cystic fibrosis (CF). It has proven to be helpful ...

Bovine Mammary Gland Biopsy Techniques

Bovine mammary gland biopsies allow researchers to collect tissue samples to study cell biology including gene expression, histological analysis, signaling pathways, and protein translation. This article describes two techniques for biopsy of the bovine mammary gland (MG). Three healthy Holstein dairy cows were the subjects. Before biopsies, cows were milked and subsequently restrained in a cattle chute. An analgesic (flunixin meglumine, 1.1 to 2.2 mg/kg of body weight) was administered via jugular intravenous [IV] injection 15-20 min prior to biopsy. For standing sedation, xylazine hydrochloride (0.01-0.05 mg/kg of body weight) was injected via the coccygeal vessels 5-10 min before the procedure. Once adequately sedated, the biopsy site was aseptically prepared and locally anaesthetized with 6 mL of 2% lidocaine hydrochloride via subcutaneous injection. Using aseptic technique, a 2 to 3 cm vertical incision was made using a number 10 scalpel. Core and needle biopsy tools were used. The core biopsy tool was attached to a cordless drill and inserted into the MG tissue through the incision using a clock-wise drill action. The needle biopsy tool was manually inserted into the incision site. Immediately after the procedure, an assistant applied pressure on the incision site for 20 to 25 min using a sterile towel to achieve hemostasis. Stainless steel surgical staples were used to oppose the skin incision. The staples were removed 10 days post-procedure. The main advantages of core and needle biopsies is that both approaches are minimally invasive procedures that can be safely performed in healthy cows. Milk yield following the biopsy was unaffected. These procedures require a short recovery time and result in fewer risks of complications. Specific limitations may include bleeding after the biopsy and infection on the biopsy site. Applications of these techniques include tissue collection for clinical diagnosis and research purposes, such as primary cell culture.

This article presents a bovine mammary gland biopsy using core and needle biopsy tools. Harvested tissue can be used for cell culture or to assess mammary physiology and metabolism including gene expression, protein expression, protein modifications, immunohistochemistry, and metabolite concentrations.

牛乳腺活检技术

Bovine mammary gland biopsies allow researchers to collect tissue samples to study cell biology including gene expression, histological analysis, ...

Applications of Spatio-temporal Mapping and Particle Analysis Techniques to Quantify Intracellular Ca2+ Signaling In Situ

Ca2+ imaging of isolated cells or specific types of cells within intact tissues often reveals complex patterns of Ca2+ signaling. This activity requires careful and in-depth analyses and quantification to capture as much information about the underlying events as possible. Spatial, temporal and intensity parameters intrinsic to Ca2+ signals such as frequency, duration, propagation, velocity and amplitude may provide some biological information required for intracellular signalling. High-resolution Ca2+ imaging typically results in the acquisition of large data files that are time consuming to process in terms of translating the imaging information into quantifiable data, and this process can be susceptible to human error and bias. Analysis of Ca2+ signals from cells in situ typically relies on simple intensity measurements from arbitrarily selected regions of interest (ROI) within a field of view (FOV). This approach ignores much of the important signaling information contained in the FOV. Thus, in order to maximize recovery of information from such high-resolution recordings obtained with Ca2+dyes or optogenetic Ca2+ imaging, appropriate spatial and temporal analysis of the Ca2+ signals is required. The protocols outlined in this paper will describe how a high volume of data can be obtained from Ca2+ imaging recordings to facilitate more complete analysis and quantification of Ca2+ signals recorded from cells using a combination of spatiotemporal map (STM)-based analysis and particle-based analysis. The protocols also describe how different patterns of Ca2+ signaling observed in different cell populations in situ can be analyzed appropriately. For illustration, the method will examine Ca2+ signaling in a specialized population of cells in the small intestine, interstitial cells of Cajal (ICC), using GECIs.

Applications of Spatio-temporal Mapping and Particle Analysis Techniques to Quantify Intracellular Ca2+ Signaling In Situ

Genetically encoded Ca2+ indicators (GECIs) have radically changed how in situ Ca2+ imaging is performed. To maximize data recovery from such recordings, appropriate analysis of Ca2+ signals is required. The protocols in this paper facilitate the quantification of Ca2+ signals recorded in situ using spatiotemporal mapping and particle-based analysis.

时空映射和粒子分析技术在细胞内 ca2 +原位信令定量中的应用

Ca2+ imaging of isolated cells or specific types of cells within intact tissues often reveals complex patterns of Ca2+ signaling. This activity requires ...

Immunohistochemistry Techniques to Analyze Cellular Proliferation and Neurogenesis in Rats Using the Thymidine Analog BrdU

One of the most important things in the field of adult hippocampal neurogenesis (AHN) is the identification of the newly generated cells. The immunodetection of thymidine analogs (such as 5-Bromo-2&#39;-deoxyuridine (BrdU)) is a standard technique used for visualizing these newly generated cells. Therefore, BrdU is usually injected in small animals intraperitoneally, so the thymidine analog gets incorporated into dividing cells during DNA synthesis. Detection is performed by immunohistochemical analysis of brain slices. Every research group that has been using this technique can appreciate that it requires special attention to minute details to achieve a successful stain. For instance, an important step is DNA denaturation with HCl, which allows it to reach the cell nucleus to stain it. However, the existing scientific reports describe very few of such steps in detail. Therefore, standardizing the technique is challenging for new laboratories as it can take several months to yield positive and successful outcomes. The purpose of this work is to describe and elaborate the steps to obtain positive and successful outcomes of the immunostaining technique in detail when working with the thymidine analog BrdU. The protocol includes the reagent preparation and setup, administration of thymidine analog in a rodent, transcardial perfusion, tissue preparation, peroxidase immunohistochemical reaction, use of avidin-biotin complex, immunofluorescence, counterstaining, microscopy imaging, and cell analysis.

This paper presents four of the most common techniques for visualizing BrdU positive cells to measure adult neurogenesis in rats. This work includes instructions for reagent preparation, thymidine analog administration, transcardial perfusion, tissue preparation, peroxidase immunohistochemical reaction, immunofluorescence, signal amplification, counterstaining, microscopy imaging, and cell analysis.