我们感谢 Dungrawala 实验室成员的意见和反馈。这项工作由 NIH 对 HD 的 R35GM137800 资助。

该方案（图 1）主要利用贴壁 U2OS 癌细胞系，但适用于在组织培养中生长的多种贴壁和悬浮细胞。本研究中使用的溶液和缓冲液详见 表 1。使用的试剂和设备列在 材料表中。
1. 准备材料
<ol><li>在实验前一天，将提供的装有低熔点琼脂糖（PBS 中 1% 低熔点琼脂糖）的瓶子放入热水浴中 5-10 分钟，以确保琼脂糖完全熔化。 注意: 请勿在微波炉中加热瓶子。</li>
	<li>熔化后，根据需要将 100 μL 琼脂糖快速分装到 1.5 mL 离心管中，并将试管储存在 4 °C 下。 固化的琼脂糖可在 4 °C 下储存长达 1 年。</li>
	<li>在实验当天，从 4 °C 储存中取出所需数量的离心管。将试管置于 42 °C 的加热块或水浴中熔化琼脂糖。 注意:在收获细胞之前，将试管置于 42 °C，以确保琼脂糖完全熔化以进行重悬（步骤 3.7）。</li>
	<li>在实验当天准备新鲜的裂解缓冲液和 TAE 缓冲液，并分别将它们储存在室温和 4 °C 下，直至准备使用。此外，在 15 mL 锥形管中分装 1x PBS，并将其放入冰箱中用于重悬步骤（步骤 3.5）。</li>
	<li>在 2 孔彗星载玻片的磨砂端标记样品标识，并将其储存在室温下直至准备使用。</li>
</ol>2. 样品的制备
<ol><li>在实验前一天，将细胞接种在 6 孔培养皿中，并在 37 °C 和 5% CO2 下培养过夜。对于 U2OS 细胞，每个样品接种大约 2-3 x 105 个细胞。 注:可能需要根据用于分析的细胞类型调整细胞数量。</li>
	<li>在实验当天，按照实验设计的条件处理细胞。作为证明复制依赖性断裂诱导的阳性对照，用 1 μM CPT 处理细胞 1 小时。CPT 是一种拓扑异构酶-1 抑制剂，以复制依赖性方式诱导双链断裂14。</li>
</ol>3. 细胞重悬和裂解
<ol><li>吸出细胞培养基并用 1x PBS 冲洗两次。</li>
	<li>向每个孔中加入 300 μL 胰蛋白酶。将培养皿在组织培养箱中孵育 2-3 分钟。 注:在胰蛋白酶中长时间孵育会在彗星分析过程中产生虚假结果。</li>
	<li>向每个孔中加入 700 μL 细胞培养基。轻轻重悬并将样品转移至 1.5 mL 离心管中。 注:建议在此步骤中对细胞进行计数，以便在步骤 3.6 中获得适当的细胞密度，以减少分析过程中重叠尾部的频率。</li>
	<li>在室温下以 2400 x g 离心细胞悬液 5 分钟。</li>
	<li>吸出培养基并将细胞重悬于 1 mL 冷的 1x PBS 中。</li>
	<li>重复步骤 3.4 和步骤 3.5 两次，并以 2 x 105 个细胞/ml 的细胞密度将细胞重悬于冷的 1x PBS 中。 注:建议在此步骤中评估细胞活力，以确保存在足够数量的活细胞。建议至少 90% 的活力。</li>
	<li>将 10 μL 细胞悬液重悬于 100 μL 等分试样的熔融琼脂糖中，并使用相同的移液器吸头，轻轻混合并将混合物均匀地涂在载玻片的一个孔中。根据需要对所有样品重复此步骤。 注:避免一次从 42 °C 水浴中取出所有等分试样，以防止琼脂糖凝固。此步骤最好通过水浴进行，并且每个样品都以交错方式重悬和铺展。避免在载玻片上铺展琼脂糖时引入气泡，因为气穴会影响电泳过程中彗尾的迁移（步骤 4.5），并在干燥步骤（步骤 5.3）中引入裂纹</li>
	<li>将载玻片在 4 °C 下避光储存 15-20 分钟，以确保琼脂糖已完全凝固（图 2A）。</li>
	<li>将载玻片放入 Coplin 罐中，并在室温下将载玻片浸入裂解缓冲液中 1 小时。</li>
	<li>用冷的 TAE 缓冲液冲洗载玻片一次，然后将载玻片浸入 4 °C 的冷 TAE 缓冲液中 30 分钟。</li>
</ol>4. 电泳
<ol><li>将电泳室设置在平坦的表面上，并使用设备边缘的四个调平旋钮调平系统。将冷袋插入底部腔室，以在电泳过程中保持溶液低温。</li>
	<li>将大约 ~850 mL 的冷 1x TAE 缓冲液倒入设备中。</li>
	<li>从 4 °C 中取出载玻片，并将它们以正确的方向放在电泳托盘上，以允许断裂的 DNA 迁移。 注意:托盘可容纳 10 个 2 孔玻片。如果处理的样品数量较少，请将玻片放在托盘的中央。如果使用奇数张玻片，请包括一个空白玻片，以便每个孔可容纳 2 张玻片。</li>
	<li>轻轻地将提供的玻片托盘覆盖物放在托盘顶部以覆盖玻片。 注:为了设备的正确操作，TAE 缓冲液的体积不应低于覆盖层的底面，也不应超过覆盖层的顶面。</li>
	<li>连接电缆后，将电源设置为 21 V 并进行电泳 （1 V/cm） 40 分钟。 注:电泳时间可能需要根据用于分析的细胞类型进行调整。未处理样品中的长尾表示运行时间过长。相反，阳性对照样品中的短尾可能反映运行时间不足。</li>
</ol>5. 染色
<ol><li>电泳完成后，从装置中取出载玻片，并在室温下将其浸入 DNA 沉淀溶液中 30 分钟。</li>
	<li>弃去 DNA 沉淀溶液，将载玻片在室温下浸入 70% 乙醇溶液中 30 分钟。</li>
	<li>丢弃 70% 乙醇溶液。取出载玻片，并在 45 °C 的杂交炉中黑暗中干燥 30 分钟（图 2B）。 注:玻片可置于黑暗干燥处过夜进行干燥步骤。</li>
	<li>将孔与 100 μL 1x SYBR Green 溶液在室温下避光孵育 30 分钟。</li>
	<li>倾斜载玻片倒出 SYBR Green 溶液，然后用无绒湿巾轻轻擦拭孔边缘以去除多余的部分。</li>
	<li>将载玻片放入抽屉中，晾干 30 分钟。</li>
	<li>继续对载玻片进行成像。</li>
</ol>6. 成像和分析
<ol><li>设置显微镜以进行成像。 注意:所有图像均使用落射荧光显微镜采集。使用具有 0.45 数值孔径的 10 倍放大倍率，曝光时间为 1/40 秒，增益为 +6dB。单色 8 位的拍摄模式采用 2 x 2 像素融合和黑平衡校正。</li>
	<li>将载玻片放在显微镜载物台上，并使用 FITC 过滤器开始成像。 注意:首先使用未经处理的样品来测试曝光时间。大多数细胞不表现出彗尾，因为 DNA 更紧凑。切换到带有 CPT 处理的细胞的孔中，并使用相同的曝光时间成像。由于断裂的 DNA 末端的迁移，将在该样品中观察到长度增加的彗星尾巴。</li>
	<li>在三个技术重复中使用相同的曝光时间，每个样品至少采集 100 颗彗星的图像。 注:盖玻片末端的彗尾往往迁移不均匀，并在样品分布中充当异常值。大多数捕获用于分析的彗星都存在于盖玻片中心附近（图 3）。</li>
	<li>手动（使用 Image J）或使用免费提供的彗星评分软件导出图像并对彗星进行评分。选择可单独区分并完全包含在视野中的彗星进行评分。重叠或部分可见的彗星不被考虑用于进一步分析（图 4）。 注意:用户可以使用以下插件在 Image J 上对彗星进行评分:https://cometbio.org/index.html。图 5 中包含使用 CometScore 软件对彗星进行评分的详细信息。</li>
	<li>将从分析中获得的尾矩绘制为描述 10-90 个百分位值的箱形图和须线图。执行正态性检验以确定是否应使用参数检验或非参数检验进行统计分析。 注意:在大多数情况下，如果仅比较两个样本，则使用 Mann-Whitney 检验应用非参数检验，如果比较三个或更多样本，则使用 Kruskal-Wallis 检验和 Dunn 多重比较。</li>
</ol>

研究 DNA 复制过程中发生的 DNA 断裂程度 

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L., Issaeva, N., Schultz, N., Helleday, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydroxyurea-stalled+replication+forks+become+progressively+inactivated+and+require+two+different+rad51-mediated+pathways+for+restart+and+repair.">Hydroxyurea-stalled replication forks become progressively inactivated and require two different rad51-mediated pathways for restart and repair.</a> Mol Cell. 37 (4), 492-502 (2010).</li><li>Sestili, P., Cantoni, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osmotically+driven+radial+diffusion+of+single-stranded+DNA+fragments+on+an+agarose+bed+as+a+convenient+measure+of+DNA+strand+scission.">Osmotically driven radial diffusion of single-stranded DNA fragments on an agarose bed as a convenient measure of DNA strand scission.</a> Free Radic Biol Med. 26 (7-8), 1019-1026 (1999).</li><li>Sestili, P., Martinelli, C., Stocchi, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fast+halo+assay:+An+improved+method+to+quantify+genomic+DNA+strand+breakage+at+the+single-cell+level.">The fast halo assay: An improved method to quantify genomic DNA strand breakage at the single-cell level.</a> Mutat Res. 607 (2), 205-214 (2006).</li><li>Gorczyca, W., Gong, J., Darzynkiewicz, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+DNA+strand+breaks+in+individual+apoptotic+cells+by+the+in+situ+terminal+deoxynucleotidyl+transferase+and+nick+translation+assays.">Detection of DNA strand breaks in individual apoptotic cells by the in situ terminal deoxynucleotidyl transferase and nick translation assays.</a> Cancer Res. 53 (8), 1945-1951 (1993).</li><li>Hanada, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+structure-specific+endonuclease+mus81+contributes+to+replication+restart+by+generating+double-strand+DNA+breaks.">The structure-specific endonuclease mus81 contributes to replication restart by generating double-strand DNA breaks.</a> Nat Struct Mol Biol. 14 (11), 1096-1104 (2007).</li></ol>

作者声明没有利益冲突。

该方案概述了一种 NC 测定法，用于评估正在进行活性复制的人类细胞中的 DNA 断裂。该方案执行起来相对简单，研究人员可以很容易地使其适应高 pH 条件进行碱性分析8。它包括两个关键步骤，如步骤 3.7 和 4.5 中所述。在步骤 3.7 中，必须将熔化的琼脂糖与细胞均匀地分布在孔中，以防止彗星在分析过程中重叠。在将细胞重悬于琼脂糖中之前，确保细胞聚集体已溶解。在涂抹琼?...

彗星检测是一种凝胶电泳方法，用于检测单细胞水平的 DNA 断裂。它依赖于开放端 DNA 断裂在电泳条件下迁移的原理，而完整的 DNA 在很大程度上保持静态。断裂的 DNA 迁移是因为断裂导致超螺旋 DNA 松弛，导致其在空间上逐渐向电泳室中的阳极重新定位，从而导致免疫荧光观察到彗星状外观 1,2。然后通过量化相对于紧凑 DNA 迁移的断裂 DNA 的数量来测量 DNA 损伤的程度。
两种最常用的彗星分析方法是碱性彗星 （AC） 测定和中性彗星 （NC） 测定。AC 测定是在变性条件下使用高碱性 pH 溶液进行的，而 NC 测定是在中性 pH 的溶液中进行的。NC 和 AC 检测都可以可靠地检测细胞核中发生的 DNA 断裂。然而，碱性版本也有利于检测碱不稳定位点3。两种形式的检测都需要使用裂解条件，以确保 DNA 在电泳前不含蛋白质。
该检测是一种检测 DNA 断裂的简单方法，为确定细胞 DNA 损伤提供了几个独特的优势。该方法相对容易设置，并且需要可以在实验室制备或从商业供应商处购买的试剂。作为一种单细胞分离技术，该检测需要很少的起始材料。值得注意的是，NC 测定可以高灵敏度测量断裂，据报道每个细胞可检测到 50 至 10,000 个断裂4。该技术的多功能性体现在其广泛的应用上，包括生态毒理学 5（ecotoxicology）、人类生物监测 6 （human biomonitoring6） 和遗传毒性研究7（genotoxicity studies）。该检测试剂盒还可以可靠地用于各种细胞类型，并适用于高通量检测8 和评估特定基因组区域的断裂9。因此，该测定法是一种快速可靠的技术，用于研究单细胞水平的断裂形成。
DNA 复制过程不断受到几种内源性和外源性压力源的挑战10,11。由于此类损伤导致的复制体停滞会导致 DNA 断裂并导致基因组不稳定性增加。DNA 断裂也经常作为复制中间体出现，以促进 DNA 修复12。此外，几种化疗药物可诱导复制依赖性双链断裂 （DSB），例如喜树碱 （CPT） 13,14,15 和 PARP 抑制剂 16,17。因此，该测定法是研究 DNA 损伤性质、评估复制叉加工以及研究 DNA 损伤剂治疗潜力的有力方法。涉及用 BrdU 标记新合成的 DNA 的测定的改编可用于研究复制相关的应激表型 18,19,20,21。值得注意的是，NC 测定是在复制背景下研究 DNA 断裂诱导的可靠方法，如涉及叉蛋白表征22,23、复制中间体分析 24,25 和基因组维护中转录-复制冲突的研究26,27 所证明的那样.在此，我们概述了一种 NC 检测方案，目的是量化人类细胞复制过程中的 DNA 断裂。有兴趣评估各种分裂人类细胞中复制应激相关损伤的研究人员可以很容易地实施该方案。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1x DPBS</td>
			<td>Gibco</td>
			<td>14190144</td>
			<td></td>
		</tr>
		<tr>
			<td>Camptothecin</td>
			<td>Selleckchem</td>
			<td>S1288</td>
			<td></td>
		</tr>
		<tr>
			<td>Comet LMAgarose (LMA)</td>
			<td>R&#38;D systems</td>
			<td>4250-050-02</td>
			<td></td>
		</tr>
		<tr>
			<td>CometAssay Electrophoresis System II</td>
			<td>R&#38;D systems</td>
			<td>4250-050-ES</td>
			<td>Includes electrophoresis tank, safety lid, cables, 2/20 wells slide trays and slide tray overlay</td>
		</tr>
		<tr>
			<td>CometScore software</td>
			<td>TriTek</td>
			<td></td>
			<td>open-sourced</td>
		</tr>
		<tr>
			<td>CometSlide</td>
			<td>R&#38;D systems</td>
			<td>4250-050-03</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM, high glucose</td>
			<td>Gibco</td>
			<td>11965092</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Gibco</td>
			<td>11320033</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO&#160;</td>
			<td>FisherSci</td>
			<td>D128-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Epifluorescence microscope</td>
			<td>Keyence</td>
			<td>BZ-X810</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum - Premium</td>
			<td>Bio-Techne</td>
			<td>S11150</td>
			<td></td>
		</tr>
		<tr>
			<td>Gibco Trypsin-EDTA (0.05%), phenol red</td>
			<td>Gibco</td>
			<td>25300054</td>
			<td></td>
		</tr>
		<tr>
			<td>GraphPad Prism 10.0</td>
			<td>GraphPad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HEK293T cells</td>
			<td>ATCC</td>
			<td>CRL-11268</td>
			<td></td>
		</tr>
		<tr>
			<td>hTERT-RPE-1 cells</td>
			<td>ATCC</td>
			<td>CRL-4000</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydroxyurea</td>
			<td>Millipore Sigma</td>
			<td>H8627</td>
			<td></td>
		</tr>
		<tr>
			<td>PARP inhibitor Olaparib</td>
			<td>Selleckchem</td>
			<td>S8096</td>
			<td></td>
		</tr>
		<tr>
			<td>PowerPac&#160;</td>
			<td>FisherSci</td>
			<td>FB300Q</td>
			<td></td>
		</tr>
		<tr>
			<td>Surface Treated Sterile Tissue Culture Plate</td>
			<td>FisherSci</td>
			<td>FB012927</td>
			<td></td>
		</tr>
		<tr>
			<td>SYBR Green I Nucleic Acid Gel Stain (10,000x)</td>
			<td>FisherSci</td>
			<td>S7567</td>
			<td></td>
		</tr>
		<tr>
			<td>U2OS cells</td>
			<td>ATCC</td>
			<td>HTB-96</td>
			<td></td>
		</tr>
		<tr>
			<td>UVP&#160;HB-1000 Hybridization Incubator</td>
			<td>FisherSci</td>
			<td>UVP95003001</td>
			<td></td>
		</tr>
	</tbody>
</table>

detection of dna breaks in dividing human cells by neutral comet assay

DNA 复制不断受到各种内源性和外源性应激源的挑战，这些应激源会损害 DNA。在基因组复制过程中遇到的此类损伤会使复制体停滞并将复制叉转化为双链断裂。如果不修复，这些有毒的 DNA 断裂会触发染色体重排，导致基因组不稳定性增加和细胞转化的可能性增加。此外，癌细胞表现出持续的复制应激，这使得靶向肿瘤细胞中的复制叉脆弱性成为化疗的一种有吸引力的策略。彗星测定是研究复制过程中 DNA 断裂的一种高度通用且强大的技术。这种凝胶电泳技术能够可靠地检测单细胞水平 DNA 断裂的诱导和修复。本文概述了一个方案，允许研究人员使用叉式停滞剂在多种细胞类型中使用有丝分裂的人类细胞中 DNA 损伤的程度。将其与自动彗星评分相结合，有助于快速分析并提高研究 DNA 断裂诱导的可靠性。

通过复制应力试剂分析断裂诱导 U2OS 细胞用 DMSO、4 mM 羟基脲 （胡） 或 100 nM CPT 处理 4 小时，并通过 NC 测定分析断裂积累（图 6A）。虽然 CPT 通过阻断拓扑异构酶 1 在 S 期诱导断裂13，但 胡 诱导的核苷酸池耗竭会阻止逐渐转化为 DSB 的复制叉28。数据表明，暴露于 胡 或 CPT 会触发 S 期的断裂诱导，这可以通过 NC 测定检测到?...

作者聚焦:揭示 SNF2L 在复制叉稳定性和基因组复制中的作用

该实验步骤旨在研究 DNA 复制过程中发生的 DNA 损伤程度。在中性条件下，可以在短时间内轻松评估 DNA 断裂的诱导。此外，该方案适用于其他细胞类型和各种复制应激试剂。

通过中性彗星测定法检测分裂人类细胞中的 DNA 断裂

DNA replication is constantly challenged by a wide variety of endogenous and exogenous stressors that can damage DNA. Such lesions encountered during ...

detection-of-dna-breaks-in-dividing-human-cells-by-neutral-comet-assay

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Research

JoVE Journal

Bioengineering

Detection of DNA Breaks in Dividing Human Cells by Neutral Comet Assay

349 次观看.  University of South Florida, Tampa. 我们实验室的主要目标是研究促进人类细胞基因组复制的机制。我们的项目侧重于了解染色质重塑剂如何在复制叉上发挥作用以促进 DNA 合成。Fork 在暴露于复制压力时会经历多个处理事件。这些事件会产生复制中间体，如果不及时修复，这些中间体会转化为有毒断裂。最近的研究表明，新生 DNA 中存在单链 DNA 间隙会导致基因组不稳定，并且是?...

视频: 作者聚焦:揭示 SNF2L 在复制叉稳定性和基因组复制中的作用

In vitro Assembly of Semi-artificial Molecular Machine and its Use for Detection of DNA Damage

Naturally occurring bio-molecular machines work in every living cell and display a variety of designs 1-6. Yet the development of artificial molecular machines centers on devices capable of directional motion, i.e. molecular motors, and on their scaled-down mechanical parts (wheels, axels, pendants etc) 7-9. This imitates the macro-machines, even though the physical properties essential for these devices, such as inertia and momentum conservation, are not usable in the nanoworld environments 10. Alternative designs, which do not follow the mechanical macromachines schemes and use mechanisms developed in the evolution of biological molecules, can take advantage of the specific conditions of the nanoworld. Besides, adapting actual biological molecules for the purposes of nano-design reduces potential dangers the nanotechnology products may pose. Here we demonstrate the assembly and application of one such bio-enabled construct, a semi-artificial molecular device which combines a naturally-occurring molecular machine with artificial components. From the enzymology point of view, our construct is a designer fluorescent enzyme-substrate complex put together to perform a specific useful function. This assembly is by definition a molecular machine, as it contains one 12. Yet, its integration with the engineered part - fluorescent dual hairpin - re-directs it to a new task of labeling DNA damage12.
Our construct assembles out of a 32-mer DNA and an enzyme vaccinia topoisomerase I (VACC TOPO). The machine then uses its own material to fabricate two fluorescently labeled detector units (Figure 1). One of the units (green fluorescence) carries VACC TOPO covalently attached to its 3'end and another unit (red fluorescence) is a free hairpin with a terminal 3'OH. The units are short-lived and quickly reassemble back into the original construct, which subsequently recleaves. In the absence of DNA breaks these two units continuously separate and religate in a cyclic manner. In tissue sections with DNA damage, the topoisomerase-carrying detector unit selectively attaches to blunt-ended DNA breaks with 5'OH (DNase II-type breaks)11,12, fluorescently labeling them. The second, enzyme-free hairpin formed after oligonucleotide cleavage, will ligate to a 5'PO4 blunt-ended break (DNase I-type breaks)11,12, if T4 DNA ligase is present in the solution 13,14 . When T4 DNA ligase is added to a tissue section or a solution containing DNA with 5'PO4 blunt-ended breaks, the ligase reacts with 5'PO4 DNA ends, forming semi-stable enzyme-DNA complexes. The blunt ended hairpins will interact with these complexes releasing ligase and covalently linking hairpins to DNA, thus labeling 5'PO4 blunt-ended DNA breaks.
This development exemplifies a new practical approach to the design of molecular machines and provides a useful sensor for detection of apoptosis and DNA damage in fixed cells and tissues.

In vitro Assembly of Semi-artificial Molecular Machine and its Use for Detection of DNA Damage

We demonstrate the assembly and application of a molecular-scale device powered by a topoisomerase protein. The construct is a bio-molecular sensor which labels two major types of DNA breaks in tissue sections by attaching two different fluorophores to their ends.

在检测DNA损伤的体外半人工分子机器，其使用的大会

Naturally occurring bio-molecular machines work in every living cell and display a variety of designs 1-6. Yet the development of artificial molecular ...

科研

生物工程

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells (hESCs) and Induced Pluripotent Stem Cells (iPSCs)

We present a method for deriving natural killer (NK) cells from undifferentiated hESCs and iPSCs using a feeder-free approach. This method gives rise to high levels of NK cells after 4 weeks culture and can undergo further 2-log expansion with artificial antigen presenting cells. hESC- and iPSC-derived NK cells developed in this system have a mature phenotype and function. The production of large numbers of genetically modifiable NK cells is applicable for both basic mechanistic as well as anti-tumor studies. Expression of firefly luciferase in hESC-derived NK cells allows a non-invasive approach to follow NK cell engraftment, distribution, and function. We also describe a dual-imaging scheme that allows separate monitoring of two different cell populations to more distinctly characterize their interactions in vivo. This method of derivation, expansion, and dual in vivo imaging provides a reliable approach for producing NK cells and their evaluation which is necessary to improve current NK cell adoptive therapies.

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells hESCs and Induced Pluripotent Stem Cells iPSCs

This protocol describes the development, expansion, and in vivo imaging of NK cells derived from hESCs and iPSCs.

开发，扩展，<em&gt;在体内</em&gt;从人类胚胎干细胞（hESCs）监测人类NK细胞和诱导多能干细胞（iPS细胞）

We present a method for deriving natural killer (NK) cells from undifferentiated hESCs and iPSCs using a feeder-free approach. This method gives rise to ...

CometChip: A High-throughput 96-Well Platform for Measuring DNA Damage in Microarrayed Human Cells

DNA damaging agents can promote aging, disease and cancer and they are ubiquitous in the environment and produced within human cells as normal cellular metabolites. Ironically, at high doses DNA damaging agents are also used to treat cancer. The ability to quantify DNA damage responses is thus critical in the public health, pharmaceutical and clinical domains. Here, we describe a novel platform that exploits microfabrication techniques to pattern cells in a fixed microarray. The &#8216;CometChip&#8217; is based upon the well-established single cell gel electrophoresis assay (a.k.a. the comet assay), which estimates the level of DNA damage by evaluating the extent of DNA migration through a matrix in an electrical field. The type of damage measured by this assay includes abasic sites, crosslinks, and strand breaks. Instead of being randomly dispersed in agarose in the traditional assay, cells are captured into an agarose microwell array by gravity. The platform also expands from the size of a standard microscope slide to a 96-well format, enabling parallel processing. Here we describe the protocols of using the chip to evaluate DNA damage caused by known genotoxic agents and the cellular repair response followed after exposure. Through the integration of biological and engineering principles, this method potentiates robust and sensitive measurements of DNA damage in human cells and provides the necessary throughput for genotoxicity testing, drug development, epidemiological studies and clinical assays.

We describe here a platform that allows comet assay detection of DNA damage with unprecedented throughput. The device patterns mammalian cells into a microarray and enables parallel processing of 96 samples. The approach facilitates analysis of base level DNA damage, exposure-induced DNA damage and DNA repair kinetics.

CometChip:一种高通量的96孔平台的微阵列人类细胞测量DNA损伤

DNA damaging agents can promote aging, disease and cancer and they are ubiquitous in the environment and produced within human cells as normal cellular ...

Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors

A label-free optical biosensor based on a nanostructured porous Si is designed for rapid capture and detection of Escherichia coli K12 bacteria, as a model microorganism. The biosensor relies on direct binding of the target bacteria cells onto its surface, while no pretreatment (e.g.&#160;by cell lysis) of the studied sample is required. A mesoporous Si thin film is used as the optical transducer element of the biosensor. Under white light illumination, the porous layer displays well-resolved Fabry-P&#233;rot fringe patterns in its reflectivity spectrum. Applying a fast Fourier transform (FFT) to reflectivity data results in a single peak. Changes in the intensity of the FFT peak are monitored. Thus, target bacteria capture onto the biosensor surface, through antibody-antigen interactions, induces measurable changes in the intensity of the FFT peaks, allowing for a &#39;real time&#39; observation of bacteria attachment.
The mesoporous Si film, fabricated by an electrochemical anodization process, is conjugated with monoclonal antibodies, specific to the target bacteria. The immobilization, immunoactivity and specificity of the antibodies are confirmed by fluorescent labeling experiments. Once the biosensor is exposed to the target bacteria, the cells are directly captured onto the antibody-modified porous Si surface. These specific capturing events result in intensity changes in the thin-film optical interference spectrum of the biosensor. We demonstrate that these biosensors can detect relatively low bacteria concentrations (detection limit of 104 cells/ml) in less than an hour.

Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors

A label-free optical biosensor for rapid bacteria detection is introduced. The biosensor is based on a nanostructured porous Si, which is designed to directly capture the target bacteria cells onto its surface. We use monoclonal antibodies, immobilized onto the porous transducer, as the capture probes. Our studies demonstrate the applicability of these biosensors for the detection of low bacterial concentrations within minutes with no prior sample processing (such as cell lysis).

光学检测<em&gt; E。大肠杆菌</em&gt;由细菌介孔硅生物传感器

A label-free optical biosensor based on a nanostructured porous Si is designed for rapid capture and detection of Escherichia coli K12 bacteria, as a ...

Quantification of Global Diastolic Function by Kinematic Modeling-based Analysis of Transmitral Flow via the Parametrized Diastolic Filling Formalism

Quantitative cardiac function assessment remains a challenge for physiologists and clinicians.&#160;Although historically invasive methods have comprised the only means available, the development of noninvasive imaging modalities (echocardiography, MRI, CT) having high temporal and spatial resolution provide a new window for quantitative diastolic function assessment. Echocardiography is the agreed upon standard for diastolic function assessment, but indexes in current clinical use merely utilize selected features of chamber dimension (M-mode) or blood/tissue motion (Doppler) waveforms without incorporating the physiologic causal determinants of the motion itself.&#160;The recognition that all left ventricles (LV) initiate filling by serving as mechanical suction pumps allows global diastolic function to be assessed based on laws of motion that apply to all chambers. What differentiates one heart from another are the parameters of the equation of motion that governs filling. Accordingly, development of the Parametrized Diastolic Filling (PDF) formalism&#160;has shown that the entire range of clinically observed early transmitral flow (Doppler E-wave) patterns are extremely well fit by the laws of damped oscillatory motion.&#160;This permits analysis of individual E-waves in accordance with a causal mechanism (recoil-initiated suction) that yields three (numerically) unique lumped parameters whose physiologic analogues are chamber stiffness (k), viscoelasticity/relaxation (c), and load (xo).&#160;The recording of transmitral flow (Doppler E-waves) is standard practice in clinical cardiology and, therefore, the echocardiographic recording method is only briefly reviewed. Our focus is on determination of the PDF parameters from routinely recorded E-wave data.&#160;As the highlighted results indicate, once the PDF parameters have been obtained from a suitable number of load varying E-waves, the investigator is free to use the parameters or construct indexes from the parameters (such as stored energy 1/2kxo2, maximum A-V pressure gradient kxo, load independent index of diastolic function, etc.) and select the aspect of physiology or pathophysiology to be quantified.

Accurate, causality-based quantification of global diastolic function has been achieved by kinematic modeling-based analysis of transmitral flow via the Parametrized Diastolic Filling (PDF) formalism. PDF generates unique stiffness, relaxation,&#160;and load parameters and elucidates &#39;new&#39; physiology while providing sensitive and specific indexes of dysfunction.

全球舒张功能通过静脉流的通过参数化舒张形式主义运动学建模为基础的定量分析

Quantitative cardiac function assessment remains a challenge for physiologists and clinicians.&#160;Although historically invasive methods have comprised ...

Automated Robotic Liquid Handling Assembly of Modular DNA Devices

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; represented by &#34;devices&#34; created as combinations of individual genetic components. However, manual assembly of such large numbers of devices is time-intensive, error-prone, and costly. The increasing sophistication and scale of synthetic biology research necessitates an efficient, reproducible way to accommodate large-scale, complex, and high throughput device construction.
Here, a DNA assembly protocol using the Type-IIS restriction endonuclease based Modular Cloning (MoClo) technique is automated on two liquid-handling robotic platforms. Automated liquid-handling robots require careful, often times tedious optimization of pipetting parameters for liquids of different viscosities (e.g. enzymes, DNA, water, buffers), as well as explicit programming to ensure correct aspiration and dispensing of DNA parts and reagents. This makes manual script writing for complex assemblies just as problematic as manual DNA assembly, and necessitates a software tool that can automate script generation. To this end, we have developed a web-based software tool, http://mocloassembly.com, for generating combinatorial DNA device libraries from basic DNA parts uploaded as Genbank files. We provide access to the tool, and an export file from our liquid handler software which includes optimized liquid classes, labware parameters, and deck layout. All DNA parts used are available through Addgene, and their digital maps can be accessed via the Boston University BDC ICE Registry. Together, these elements provide a foundation for other organizations to automate modular cloning experiments and similar protocols.
The automated DNA assembly workflow presented here enables the repeatable, automated, high-throughput production of DNA devices, and reduces the risk of human error arising from repetitive manual pipetting. Sequencing data show the automated DNA assembly reactions generated from this workflow are ~95% correct and require as little as 4% as much hands-on time, compared to manual reaction preparation.

Here, an automated workflow to perform modular DNA &#34;device&#34; assembly using a modular cloning DNA assembly method on liquid-handling robots is presented. The protocol uses an intuitive software tool for generating liquid handler picklists for combinatorial DNA device library generation, which we demonstrate using two liquid handling platforms.

模块化 DNA 设备的自动机器人液体处理组件

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.

用利穆鲁布莱巴细胞裂解液 (lal) 法检测纳米配方中的内毒素

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause ...

Uptake of New Lipid-coated Nanoparticles Containing Falcarindiol by Human Mesenchymal Stem Cells

Nanoparticles are the focus of an increased interest in drug delivery systems for cancer therapy. Lipid-coated nanoparticles are inspired in structure and size by low-density lipoproteins (LDLs) because cancer cells have an increased need for cholesterol to proliferate, and this has been exploited as a mechanism for delivering anticancer drugs to cancer cells. Moreover, depending on drug chemistry, encapsulating the drug can be advantageous to avoid degradation of the drug during circulation in vivo. Therefore, in this&#160;study, this design is used to fabricate lipid-coated nanoparticles of the anticancer drug falcarindiol, providing a potential new delivery system of falcarindiol in order to stabilize its chemical structure against degradation and improve its uptake by tumors. Falcarindiol nanoparticles, with a phospholipid and cholesterol monolayer encapsulating the purified drug core of the particle, were designed. The lipid monolayer coating consists of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol (Chol), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE PEG 2000) along with the fluorescent label DiI (molar ratios of 43:50:5:2). The nanoparticles are fabricated using the rapid injection method, which is a fast and simple technique to precipitate nanoparticles by good-solvent for anti-solvent exchange. It consists of a rapid injection of an ethanol solution containing the nanoparticle components into an aqueous phase. The size of the fluorescent nanoparticles is measured using dynamic light scattering (DLS) at 74.1 &#177; 6.7 nm. The uptake of the nanoparticles is tested in human mesenchymal stem cells (hMSCs) and imaged using fluorescence and confocal microscopy. The uptake of the nanoparticles is observed in hMSCs, suggesting the potential for such a stable drug delivery system for falcarindiol.

This article describes the encapsulation of falcarindiol in lipid-coated 74 nm nanoparticles. The cellular uptake of the nanoparticles&#160;by human stem cells into lipid droplets is monitored by fluorescent and confocal imaging. Nanoparticles are fabricated by the rapid injection method of solvent shifting, and their size is measured with the dynamic light scattering technique.

人骨髓间充质干细胞吸收含有法卡林多醇的新型脂质包覆纳米颗粒

Nanoparticles are the focus of an increased interest in drug delivery systems for cancer therapy. Lipid-coated nanoparticles are inspired in structure and ...

Scalable Generation of Mature Cerebellar Organoids from Human Pluripotent Stem Cells and Characterization by Immunostaining

The cerebellum plays a critical role in the maintenance of balance and motor coordination, and a functional defect in different cerebellar neurons can trigger cerebellar dysfunction. Most of the current knowledge about disease-related neuronal phenotypes is based on postmortem tissues, which makes understanding of disease progression and development difficult. Animal models and immortalized cell lines have also been used as models for neurodegenerative disorders. However, they do not fully recapitulate human disease. Human induced pluripotent stem cells (iPSCs) have great potential for disease modeling and provide a valuable source for regenerative approaches. In recent years, the generation of cerebral organoids from patient-derived iPSCs improved the prospects for neurodegenerative disease modeling. However, protocols that produce large numbers of organoids and a high yield of mature neurons in 3D culture systems are lacking. The protocol presented is a new approach for reproducible and scalable generation of human iPSC-derived organoids under chemically-defined conditions using scalable single-use bioreactors, in which organoids acquire cerebellar identity. The generated organoids are characterized by the expression of specific markers at both mRNA and protein level. The analysis of specific groups of proteins allows the detection of different cerebellar cell populations, whose localization is important for the evaluation of organoid structure. Organoid cryosectioning and further immunostaining of organoid slices are used to evaluate the presence of specific cerebellar cell populations and their spatial organization.

This protocol describes a dynamic culture system to produce controlled size aggregates of human pluripotent stem cells and further stimulate differentiation in cerebellar organoids under chemically-defined and feeder-free conditions using a single-use bioreactor.

人类多能干细胞和免疫损伤的成熟小脑有机体可伸缩生成

The cerebellum plays a critical role in the maintenance of balance and motor coordination, and a functional defect in different cerebellar neurons can ...

Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically carried out in low throughput. Rapid and ongoing expansion of induced pluripotent stem cell (iPSC) technology presents a new standard in cardiovascular research and alternate methods are now necessary to increase throughput of electrophysiological data at a single cell level. VF2.1Cl is a recently derived voltage sensitive dye which provides a rapid single channel, high magnitude response to fluctuations in membrane potential. It possesses kinetics superior to those of other existing voltage indicators and makes available functional data equivalent to that of traditional microelectrode techniques. Here, we demonstrate simplified, non-invasive action potential characterization in externally paced human iPSC derived cardiomyocytes using a modular and highly affordable photometry system.

Here we describe optical acquisition and characterization of action potentials from induced pluripotent stem cell derived cardiomyocytes using a high-speed modular photometry system.

人类诱导多能干细胞衍生心肌细胞中的单细胞光学作用潜在测量

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically ...