我们要感谢Prachi Shah女士最初帮助我们优化协议。我们要感谢Manoj Garg博士提供A2780卵巢癌细胞系。EK得到了生物技术系（编号BT / RLF / Re-entry/06 / 2015），科学和技术部（ECR / 2018 / 002117）和NMIMS种子基金（IO 401405）的研究资助。

注意：有关以下方案中使用的所有试剂的详细信息，请参阅 材料表 。表 1 列出了实验室制造的试剂以及优化的体积， 表2 显示了市售试剂的工作浓度。
1. 细胞培养
<ol><li>在 Dulbecco 的改良鹰培养基 （DMEM） 完全培养基中维持要测量端粒长度的细胞（此处使用的是 A2780 细胞，这是一种卵巢腺癌细胞系），该培养基补充有 10% 胎牛血清 （FBS）、链霉素、青霉素和两性霉素 B 在 6 cm 培养皿中。在含有5%二氧化碳的加湿和受控环境中于37°C孵育，直到细胞80%-100%汇合。</li>
	<li>取出培养基并用 5 mL 的 1x 磷酸盐缓冲盐水 （PBS） 洗涤。</li>
	<li>用1mL胰蛋白酶 - 乙二胺四乙酸（EDTA）处理细胞，通过在37°C孵育3-5分钟来分离细胞。</li>
	<li>加入 2 mL DMEM 完全培养基以灭活胰蛋白酶并将细胞收集在离心管中。</li>
	<li>以2，348× g 沉淀细胞5分钟。</li>
	<li>用1x PBS洗涤沉淀，并以2，348× g 离心5分钟。</li>
	<li>将颗粒储存在-80°C直至进一步使用。 注意：细胞计数可能因细胞系而异。在A2780细胞系的情况下，我们获得了大约2.5×106 个细胞，用于进一步的步骤。</li>
</ol>2. 基因组DNA分离
<ol><li>向细胞沉淀中加入 500 μL 裂解缓冲液（10 mM tris-Cl，pH 8.0;25 mM EDTA，pH 8.0;100 mM NaCl;0.5% w/v 十二烷基硫酸钠），并使用切割尖端（开口直径至少为 2 mm）轻轻混合。加入 20 μg/mL 新鲜制备的 RNase A，并通过倒置轻轻混合。</li>
	<li>在37°C孵育30分钟，并在孵育过程中偶尔倒置管。</li>
	<li>加入蛋白酶K至终浓度为100μg/ mL，并通过倒置轻轻混合10次。在55°C孵育2小时。在孵育期间定期（每10分钟）倒置管。</li>
	<li>加入 500 μL 苯酚：氯仿：异戊醇试剂 （25：24：1），倒置轻轻混合 20 次。在室温（约25°C）下以9，391 ×g 离心15分钟。 注意： 图1A 显示了离心后获得的三层。</li>
	<li>从可见的三层中取出粘稠的上层水层，使用切口放入新管中，并加入等量的氯仿。轻轻倒置混合20次。</li>
	<li>在室温下以9，391 ×g 离心管15分钟。</li>
	<li>收集水层并加入适量的5M NaCl，使NaCl的最终浓度为0.2 M。</li>
	<li>加入两体积的100%乙醇。轻轻倒置混合20-25次。在室温下以15，871× g 离心5分钟。</li>
	<li>除去上清液并加入 500 μL 70% 乙醇以洗涤沉淀。在室温下以15，871× g 离心5分钟。</li>
	<li>除去上清液，风干沉淀几分钟，然后加入 50 μL 无菌无核酸酶水。让DNA在室温下再水化1-2天。使用切头移液混合。</li>
	<li>使用紫外（UV）分光光度法测量DNA的浓度。如有必要，使用无菌无核酸酶水重新稀释样品，以获得最低浓度为 300-500 ng/μL。</li>
	<li>通过在1%琼脂糖凝胶上运行来检查DNA的完整性（图1B）。</li>
	<li>将稀释的DNA储存在-20°C直至进一步使用。</li>
</ol>3. 基因组DNA的消化
<ol><li>制备Rsa1（20U）和Hinf1（20U）的酶混合物，每次反应加入2μL限制性酶切缓冲液。</li>
	<li>取适当体积的基因组DNA，使DNA总量为1.5μg。用无菌无核酸酶的水补足体积，使酶加入后的总体积为20μL。</li>
	<li>通过敲击混合均匀，然后脉冲旋转。</li>
	<li>将混合物在37°C孵育2小时。</li>
</ol>4. 琼脂糖凝胶电泳
<ol><li>使用高级琼脂糖在 1x 三乙酸酯 EDTA （TAE） 缓冲液中制备 10 cm × 15 cm 0.8% 琼脂糖凝胶。</li>
	<li>向每个消化的基因组 DNA 样品中加入 5 μL 上样染料，从而使最终体积为 25 μL，并将样品上样到凝胶上。</li>
	<li>使用 1 μL 分子标记物（分子量标准）、3 μL 无菌无核酸酶水和 1 μL 5x 上样染料制备分子标记混合物。</li>
	<li>如果样品数量较多（~10），则在基因组DNA消化样品的两侧加载5 μL分子标记混合物，如果样品少于或等于5个，则仅在一侧加载。</li>
	<li>以5 V / cm运行凝胶6小时。电泳完成后，在凝胶的一角做一个凹口以标记上样顺序。</li>
	<li>将凝胶浸入0.25M HCl中，在室温下轻轻搅拌10分钟。</li>
	<li>用蒸馏水冲洗凝胶两次。</li>
	<li>通过将凝胶浸入0.5M NaOH和1.5M NaCl的溶液中两次，每次15分钟，在室温下轻轻搅拌，使DNA变性。</li>
	<li>用蒸馏水冲洗凝胶两次。</li>
	<li>通过将凝胶浸入 0.5 M tris-HCl 和 3 M NaCl （pH 7.5） 的溶液中两次，在室温下轻轻搅拌 15 分钟来中和 DNA。</li>
</ol>5. 南方印迹
<ol><li>切下10厘米×12厘米大小的尼龙膜，并在与凝胶相同的位置做一个缺口。</li>
	<li>通过浸入蒸馏水，然后浸入20x盐水柠檬酸钠（SSC）缓冲液来激活膜（见 表1）。</li>
	<li>设置转移。<ol><li>拿一个干净的玻璃托盘，将另一个托盘倒置放入其中。使用普通滤纸制作灯芯，使两端接触外托盘的底部。</li>
		<li>将凝胶放在滤纸上。轻轻地将活化的尼龙膜放在凝胶上，凹口重叠，并通过滚动玻璃棒去除任何气泡。</li>
		<li>将 2 厘米的 9.5 厘米堆× 11.5 厘米纤维素滤纸，然后放入一堆 6 厘米相同尺寸的普通滤纸。在此设置顶部放置一个砝码，以便在下方具有相等的权重分布。用20x SSC缓冲液填充外托盘（见 图2）。</li>
		<li>将设置留给隔夜转移。</li>
	</ol></li>
	<li>南方转移后，通过在紫外透射仪或紫外交联器上将转移的DNA固定在膜上。使用 302 nm 8 W 灯 5 分钟，或 254 nm 8 W 灯 2 分钟，相当于大约 120 mJ。确保转移DNA的膜侧面向灯。</li>
	<li>用 25 mL 的 2x SSC 缓冲液洗涤膜两次。</li>
	<li>如果需要，通过完全干燥印迹并将其储存在2-8°C后将其松散地包裹在箔纸中来暂停方案，直到进一步处理。</li>
</ol>6. 杂交和化学发光检测
<ol><li>将预杂交缓冲液预热至42°C。 注意：以下步骤包括每100 cm2 印迹膜要使用的溶液/缓冲液体积。</li>
	<li>将膜在10mL预杂交缓冲液中在42°C下孵育1小时，轻轻搅拌以进行预杂交。</li>
	<li>每 5 mL 预热预杂交缓冲液加入 0.5 μL 端粒探针，制成杂交溶液。</li>
	<li>将印迹在42°C的10mL杂交溶液中孵育3小时，轻轻搅拌。</li>
	<li>在室温下用严格的缓冲液1（表1）洗涤印迹两次，每次10分钟（每次25mL）。</li>
	<li>在50°C下预热严格的缓冲液2（表1）30分钟。</li>
	<li>用严格的缓冲液2洗涤印迹两次，在50°C下轻轻搅拌15分钟（每次25mL）。</li>
	<li>用 15 mL 洗涤缓冲液冲洗 5 分钟，在室温下轻轻搅拌。</li>
	<li>将膜在室温下在10mL新鲜制备的1x封闭溶液中孵育30分钟，轻轻搅拌。</li>
	<li>将膜在10mL与碱性磷酸酶工作溶液（见 表2）偶联的抗地灰辛中孵育30分钟，并在室温下轻轻搅拌。</li>
	<li>用洗涤缓冲液在室温下轻轻搅拌两次印迹15分钟。</li>
	<li>在室温下在 10 mL 检测缓冲液中孵育 5 分钟，轻轻搅拌。</li>
	<li>去除多余的检测缓冲液，并在杂交袋或乙酸盐片上保持DNA朝上的膜。将~1-1.5mL底物溶液滴加到膜上，立即将另一张纸放在其上，并在室温下孵育5分钟。</li>
	<li>挤出多余的底物溶液进行成像。</li>
	<li>在凝胶成像系统中对印迹膜成像约20分钟，在不同时间点收集多个图像。选择不饱和的图像进行进一步分析。 注意：如果信号较弱，可以进行更长的成像时间。</li>
</ol>7. 分析
<ol><li>获取图像文件后，以尽可能高的分辨率（在本例中为 600 dpi）以.tif格式导出它们以供发布。</li>
	<li>安装 端工具 软件。这需要运行特定版本的 MATLAB（免费提供）（R2012a）。</li>
	<li>打开软件，然后单击右上角的 “加载图像文件” 按钮。</li>
	<li>加载图像后，单击反转图像，使 图像背景为黑色，拖尾/条带为白色。</li>
	<li>从孔开始裁剪顶角的图像。选择裁剪后，右键单击其中并选择 裁剪图像。</li>
	<li>裁剪图像后，单击计算车道并允许自动进行 车道 检测。</li>
	<li>如果未正确检测到车道，例如，根本没有检测到某些车道，或者检测到额外或部分车道，请按如下所述执行车道调整。<ol><li>单击调整车道，然后在打开的弹出窗口中根据需要添加和/或调整 车道 ，然后单击 应用并关闭。</li>
	</ol></li>
	<li>调整车道后，确保在每个车道上看到一个红点，并使用梯子 拟合 按钮调整梯子，确保两个梯形车道中的峰数位于同一位置。使用 “删除极值 ”按钮删除任何无关的峰值。</li>
	<li>调整梯形图后，选择“ 车道剖面图”，单击 “梯形拟合”，然后选择“多项 式拟合”。</li>
	<li>单击软件建议的趋势 线，然后在下一个选项中选择 更正 。</li>
	<li>单击 “获取 结果”以表格形式显示结果，其中 平均 TRF 行是相应泳道样本的端粒长度测量值。</li>
	<li>单击 全部保存 以电子表格的形式存储结果。</li>
</ol>

使用非放射性化学发光检测定量端粒长度

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作者没有利益冲突。

我们描述了一种使用南方印迹法测量端粒长度的非放射性、基于化学发光的方法的详细程序。该方案已经过测试，允许明智地使用多种试剂，而不会影响结果质量。预杂交和杂交缓冲液最多可重复使用五次。酶浓度可以在每1.5-2μg基因组DNA的10-20U之间变化，而不会影响结果。其他几种试剂盒组分，如DIG标记的分子量标记物和杂交探针，可以在低于推荐浓度的浓度下使用。优化的卷如 表 2</stro...

端粒是存在于染色体末端的重复DNA序列。它们具有TTAGGG的串联重复，并通过保护染色体免受磨损和末端复制问题来维持基因组完整性，这意味着3'突出的部分无法被DNA聚合酶1，2复制。短端粒导致细胞染色体异常，因此细胞在称为复制衰老3的阶段永久停滞。短端粒还会导致许多其他问题，例如线粒体功能障碍4，5 和细胞功能障碍。
当细胞分裂时，DNA端粒重复序列丢失，平均每年损失25至200 bp6，导致细胞在一定次数的分裂后衰老6。衰老与合并症的频率较高有关，其特征是端粒长度缩短7。正如Mender所描述的端粒限制性片段（TRF）分析是一种非常昂贵的方法8。因此，在大多数研究中，在量化端粒长度时没有实施。
目前，大多数流行病学研究采用基于定量聚合酶链反应（qPCR）的端粒长度测量。然而，基于qPCR的方法是一种相对测量方法，因为它测量端粒和单拷贝基因扩增产物之间的比率，而不是绝对端粒长度。使用 TRF 协议测量端粒长度是金标准方法，因为它可以测量样品中的端粒长度分布，并且测量值可以用以千碱基 （kb） 为单位的绝对值表示。然而，它的使用是有限的，因为它繁琐、劳动密集型且成本高昂。在这里，我们提出了一种使用基于化学发光的TRF测量端粒长度的优化协议。
TRF 分析包括七个主要步骤：1） 培养用于基因组 DNA 提取的细胞，2） 使用苯酚：氯仿：异戊醇 （P：C：I） 方法进行基因组 DNA 提取，3） 基因组 DNA 的限制性酶切，4） 琼脂糖凝胶电泳，5） 限制性酶切 DNA 片段的南部印迹，6） 杂 交和检测化学发光 - 固定化端粒探针通过碱性磷酸酶的高灵敏度化学发光底物可视化，2-氯-5-（4-甲氧基螺[1，2-二氧杂环丁烷-3，2′-（5-氯三环[3.3.1.13.7]癸烷]）-4-基]-1-苯基磷酸酯（CDP-Star）-和7）分析，以从这些端粒涂片中获得平均端粒长度和范围信息。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Cell Line</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>A2780 (Ovarian adenocarcinoma cell line)</td>
			<td>Received as a gift</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ChemiDoc XRS+ (for imaging and UV cross linking)</td>
			<td>Biorad</td>
			<td>Universal hood II (721BR14277)</td>
			<td></td>
		</tr>
		<tr>
			<td>Nanodrop (Epoch 2)</td>
			<td>Biotek</td>
			<td>EPOCH2</td>
			<td></td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TeloTool</td>
			<td>Version 1.3</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic Acid</td>
			<td>Molychem</td>
			<td>64-19-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>MP</td>
			<td>180720</td>
			<td></td>
		</tr>
		<tr>
			<td>Amphotericin B</td>
			<td>Gibco, ThermoFisher Scientific, USA</td>
			<td>15240062</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM&#160;</td>
			<td>HyClone, Cytiva, USA</td>
			<td>SH30243.01</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediamine tetraacetic acid&#160;</td>
			<td>Molychem</td>
			<td>6381-92-6</td>
			<td></td>
		</tr>
		<tr>
			<td>HI FBS</td>
			<td>Gibco, ThermoFisher Scientific, USA</td>
			<td>10270106</td>
			<td></td>
		</tr>
		<tr>
			<td>HCl</td>
			<td>Molychem</td>
			<td>76-47-01-0</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Molychem</td>
			<td>7647-14-5</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Molychem</td>
			<td>1310-73-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Nylon membrane</td>
			<td>Sigma</td>
			<td>11209299001</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin</td>
			<td>Gibco, ThermoFisher Scientific, USA</td>
			<td>15240062</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulfate</td>
			<td>Affymetrix</td>
			<td>151-21-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptomycin</td>
			<td>Gibco, ThermoFisher Scientific, USA</td>
			<td>15240062</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>BIORAD</td>
			<td>77-86-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris HCl</td>
			<td>Sigma Aldrich</td>
			<td>1185-53-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Whatman paper</td>
			<td>GE healthcare lifesciences</td>
			<td>1001-917</td>
			<td></td>
		</tr>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1 kb ladder</td>
			<td>NEB</td>
			<td>N3232S</td>
			<td></td>
		</tr>
		<tr>
			<td>20x SSC</td>
			<td>Invitrogen</td>
			<td>15557-036</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti DIG AP</td>
			<td>Telo TAGGG Telomere Length Assay kit</td>
			<td>12209136001</td>
			<td></td>
		</tr>
		<tr>
			<td>Blocking solution 10x</td>
			<td>Telo TAGGG Telomere Length Assay kit</td>
			<td>12209136001</td>
			<td></td>
		</tr>
		<tr>
			<td>Cutsmart Buffer</td>
			<td>NEB</td>
			<td>B6004</td>
			<td></td>
		</tr>
		<tr>
			<td>Detection buffer 10x</td>
			<td>Telo TAGGG Telomere Length Assay kit</td>
			<td>12209136001</td>
			<td></td>
		</tr>
		<tr>
			<td>Dig easy hyb</td>
			<td>Telo TAGGG Telomere Length Assay kit</td>
			<td>12209136001</td>
			<td></td>
		</tr>
		<tr>
			<td>Digestion Buffer</td>
			<td>Telo TAGGG Telomere Length Assay kit</td>
			<td>12209136001</td>
			<td></td>
		</tr>
		<tr>
			<td>Hinf 1</td>
			<td>Telo TAGGG Telomere Length Assay kit</td>
			<td>12209136001</td>
			<td></td>
		</tr>
		<tr>
			<td>Hinf 1 (alternative to kit)</td>
			<td>NEB</td>
			<td>R0155T</td>
			<td></td>
		</tr>
		<tr>
			<td>Loading Dye</td>
			<td>BIOLABS</td>
			<td>N3231S</td>
			<td></td>
		</tr>
		<tr>
			<td>Maleic acid buffer 10x</td>
			<td>Telo TAGGG Telomere Length Assay kit</td>
			<td>12209136001</td>
			<td></td>
		</tr>
		<tr>
			<td>Molecular marker</td>
			<td>Telo TAGGG Telomere Length Assay kit</td>
			<td>12209136001</td>
			<td></td>
		</tr>
		<tr>
			<td>Probe</td>
			<td>Telo TAGGG Telomere Length Assay kit</td>
			<td>12209136001</td>
			<td></td>
		</tr>
		<tr>
			<td>Rsa 1</td>
			<td>Telo TAGGG Telomere Length Assay kit</td>
			<td>12209136001</td>
			<td></td>
		</tr>
		<tr>
			<td>Rsa 1 (alternative to kit)</td>
			<td>NEB</td>
			<td>R0167L</td>
			<td></td>
		</tr>
		<tr>
			<td>Substrate</td>
			<td>Telo TAGGG Telomere Length Assay kit</td>
			<td>12209136001</td>
			<td></td>
		</tr>
		<tr>
			<td>Wash buffer</td>
			<td>Telo TAGGG Telomere Length Assay kit</td>
			<td>12209136001</td>
			<td></td>
		</tr>
	</tbody>
</table>

optimization of performance parameters of the taggg telomere length assay

端粒是存在于染色体末端的重复序列;它们的缩短是人类体细胞的特征。缩短是由于末端复制的问题和端粒酶的缺失而发生的，端粒酶负责维持端粒长度。有趣的是，端粒也会响应各种内部生理过程而缩短，如氧化应激和炎症，这些过程可能由于污染物、传染因子、营养物质或辐射等细胞外因素而受到影响。因此，端粒长度是衰老和各种生理健康参数的极好生物标志物。TAGGG 端粒长度测定试剂盒用于使用端粒限制性片段 （TRF） 测定来量化平均端粒长度，并且具有高度可重复性。然而，这是一种昂贵的方法，因此，它不常规用于大样本数量。在这里，我们描述了一个详细的方案，用于使用南方印迹或TRF分析以及基于非放射性化学发光的检测来优化且经济高效地测量端粒长度。

在1%琼脂糖凝胶上运行的提取的基因组DNA（gDNA）显示出良好的完整性，如图 1B所示，表明该样品可用于TRF的进一步下游处理。然后通过修改每个步骤所需的溶液体积来进行TRF测定（见表 1 和 表2）。TRF 信号清晰可见（图 3）。因此，通过修改溶液体积和浓度，可以处理更多的样品而不会对结果产生任何负面影响，并且可以使用?...

Watch this Scientific Journal Video about Optimization of Performance Parameters of the TAGGG Telomere Length Assay at JoVE.com

作者聚焦：TAGGG 端粒长度测定性能参数的优化  

在这里，我们详细描述了使用非放射性化学发光检测量化端粒长度的方案，重点是优化TAGGG端粒长度测定试剂盒的各种性能参数，例如缓冲液量和探针浓度。

TAGGG端粒长度测定性能参数优化

Telomeres are repetitive sequences which are present at chromosomal ends; their shortening is a characteristic feature of human somatic cells. Shortening ...

我们的研究范围涉及测量不同研究组的端粒长度差异。我们试图回答的问题围绕着生理疾病的疾病以及它们如何影响端粒长度，反之亦然。该技术目前用于推进端粒长度生物学领域的研究，包括纳米孔测序和最近基于CRISPR Cas的测量。目前的实验挑战是准确测量绝对端粒长度及其变化。当使用不同的方法来测量端粒长度时，同一样品可能会产生不同的结果。我们解决的研究差距是缺乏测量端粒长度的标准方法。端粒限制性片段分析（TRF）仍然是评估所有新技术的标准。此外，我们优化的实验方案有助于以有限的资源处理更多的样品。我们的研究结果将有助于推进端粒长度生物学领域的研究，提供一种可靠且相对经济的方法来将新方法与黄金标准（TRF）进行比较。我们的实验室将专注于小分子抑制剂对端粒长度的影响。我们还将研究生活方式疾病和端粒长度对彼此的影响。

optimization-performance-parameters-taggg-telomere-length

Research

JoVE Journal

Cancer Research

Optimization of Performance Parameters of the TAGGG Telomere Length Assay

1.6K 次观看.  SVKM's NMIMS Deemed-to-be-University. 我们的研究范围涉及测量不同研究组的端粒长度差异。我们试图回答的问题围绕着生理疾病的疾病以及它们如何影响端粒长度，反之亦然。该技术目前用于推进端粒长度生物学领域的研究，包括纳米孔测序和最近基于CRISPR Cas的测量。目前的实验挑战是准确测量绝对端粒长度及其变化。当使用不同的方法来测量端粒长度时，同一样品可能会产生不同的结果。我们解决的研...

视频: 作者聚焦：TAGGG 端粒长度测定性能参数的优化  

A Comprehensive Procedure to Evaluate the In Vivo Performance of Cancer Nanomedicines

Inspired by the success of previous cancer nanomedicines in the clinic, researchers have generated a large number of novel formulations in the past decade. However, only a small number of nanomedicines have been approved for clinical use, whereas the majority of nanomedicines under clinical development have produced disappointing results. One major obstacle to the successful clinical translation of new cancer nanomedicines is the lack of an accurate understanding of their in vivo performance. This article features a rigorous procedure to characterize the in vivo behavior of nanomedicines in tumor-bearing mice at systemic, tissue, single-cell, and subcellular levels via the integration of positron emission tomography-computed tomography (PET-CT), radioactivity quantification methods, flow cytometry, and fluorescence microscopy. Using this approach, researchers can accurately evaluate novel nanoscale formulations in relevant mouse models of cancer. These protocols may have the ability to identify the most promising cancer nanomedicines with high translational potential or to aid in the optimization of cancer nanomedicines for future translation.

A Comprehensive Procedure to Evaluate the In Vivo Performance of Cancer Nanomedicines

The poor understanding of the in vivo performance of nanomedicines stymies their clinical translation. Procedures to evaluate the in vivo behavior of cancer nanomedicines at systemic, tissue, single-cell, and subcellular levels in tumor-bearing immunocompetent mice are described here. This approach may help researchers to identify promising cancer nanomedicines for clinical translation.

综合程序，以评估<em&gt;在体内</em&gt;癌症纳米医学的性能

Inspired by the success of previous cancer nanomedicines in the clinic, researchers have generated a large number of novel formulations in the past ...

科研

癌症研究

Modified Terminal Restriction Fragment Analysis for Quantifying Telomere Length Using In-gel Hybridization

There are several different techniques for measuring telomere length, each with their own advantages and disadvantages. The traditional approach, Telomere Restriction Fragment (TRF) analysis, utilizes a DNA hybridization technique whereby genomic DNA samples are digested with restriction enzymes, leaving behind telomere DNA repeats and some sub-telomeric DNA. These are separated by agarose gel electrophoresis, transferred to a filter membrane and hybridized to oligonucleotide probes tagged with either chemiluminescence or radioactivity to visualize telomere restriction fragments. This approach, while requiring a larger quantity of DNA than other techniques such as PCR, can measure the telomere length distribution of a population of cells and allows measurement expressed in absolute kilobases. This manuscript demonstrates a modified DNA hybridization procedure for determining telomere length. Genomic DNA is first digested with restriction enzymes (that do not cut telomeres) and separated by agarose gel electrophoresis. The gel is then dried and the DNA is denatured and hybridized in situ to a radiolabeled oligonucleotide probe. This in situ hybridization avoids loss of telomere DNA and improves signal intensity. Following hybridization, the gels are imaged utilizing phosphor screens and the telomere length is quantified using a graphing program. This procedure was developed by the laboratories of Drs. Woodring Wright and Jerry Shay at the University of Texas Southwestern1,2. Here, we present a detailed description of this procedure, with some modifications.

In this article, a detailed protocol for quantifying telomere length using a modified terminal restriction fragment analysis is discussed that provides fast and efficient direct measurement of telomere length. This technique can be applied to a variety of cell sources of DNA for quantifying telomere length.

使用凝胶内杂交来定量端粒长度的修饰末端限制片段分析

There are several different techniques for measuring telomere length, each with their own advantages and disadvantages. The traditional approach, Telomere ...

Evaluation of the Cell Invasion and Migration Process: A Comparison of the Video Microscope-based Scratch Wound Assay and the Boyden Chamber Assay

The invasion and migration abilities of tumor cells are main contributors to cancer progression and recurrence. Many studies have explored the migration and invasion abilities to understand how cancer cells disseminate, with the aim of developing new treatment strategies. Analysis of the cellular and molecular basis of these abilities has led to the characterization of cell mobility and the physicochemical properties of the cytoskeleton and cellular microenvironment. For many years, the Boyden chamber assay and the scratch wound assay have been the standard techniques to study cell invasion and migration. However, these two techniques have limitations. The Boyden chamber assay is difficult and time consuming, and the scratch wound assay has low reproducibility. Development of modern technologies, especially in microscopy, has increased the reproducibility of the scratch wound assay. Using powerful analysis systems, an &#34;in-incubator&#34; video microscope can be used to provide automatic and real-time analysis of cell migration and invasion. The aim of this paper is to report and compare the two assays used to study cell invasion and migration: the Boyden chamber assay and an optimized in vitro video microscope-based scratch wound assay.

This study reports two different methods for the analysis of cell invasion and migration: the Boyden chamber assay and the in vitro video microscope-based wound-healing assay. The protocols for these two experiments are described, and their benefits and disadvantages are compared.

细胞侵袭和迁移过程的评价: 视频 Microscope-based 刮伤法与博伊登室法的比较

The invasion and migration abilities of tumor cells are main contributors to cancer progression and recurrence. Many studies have explored the migration ...

Using the Dot Assay to Analyze Migration of Cell Sheets

Although complex organisms appear static, their tissues are under a continuous turnover. As cells age, die, and are replaced by new cells, cells move within tissues in a tightly orchestrated manner. During tumor development, this equilibrium is disturbed, and tumor cells leave the epithelium of origin to invade the local microenvironment, to travel to distant sites, and to ultimately form metastatic tumors at distant sites. The dot assay is a simple, two-dimensional unconstrained migration assay, to assess the net movement of cell sheets into a cell-free area, and to analyze parameters of cell migration using time-lapse imaging. Here, the dot assay is demonstrated using a human invasive, lung colony forming breast cancer cell line, MCF10CA1a, to analyze the cells' migratory response to epidermal growth factor (EGF), which is known to increase malignant potential of breast cancer cells and to alter the migratory phenotype of cells.

Cell migration is essential for development, tissue maintenance and repair, and tumorigenesis, and is regulated by growth factors, chemokines, and cytokines. This protocol describes the dot assay, a two-dimensional, unconstrained migration assay to assess the migratory phenotype of attached, cohesive cell sheets in response to microenvironmental cues.

用点法分析细胞板的迁移

Although complex organisms appear static, their tissues are under a continuous turnover. As cells age, die, and are replaced by new cells, cells move ...

A Comprehensive Procedure to Evaluate the In Vitro Performance of the Putative Hemangioblastoma Neovascularization Using the Spheroid Sprouting Assay

The inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene plays a crucial role in the development of hemangioblastomas (HBs) within the human central nervous system (CNS). However, both the cytological origin and the evolutionary process of HBs (including neovascularization) remain controversial, and anti-angiogenesis for VHL-HBs, based on classic HB angiogenesis, have produced disappointing results in clinical trials. One major obstacle to the successful clinical translation of anti-vascular treatment is the lack of a thorough understanding of neovascularization in this vascular tumor. In this article, we present a comprehensive procedure to evaluate in vitro whether classic tumor angiogenesis exists in HBs, as well as its role in HBs. With this procedure, researchers can accurately understand the complexity of HB neovascularization and identify the function of this common form of angiogenesis in HBs. These protocols can be used to evaluate the most promising anti-vascular therapy for tumors, which has high translational potential either for tumors treatment or for aiding in the optimization of the anti-angiogenic treatment for HBs in future translations. The results highlight the complexity of HB neovascularization and suggest that this common form angiogenesis is only a complementary mechanism in HB neovascularization.

A Comprehensive Procedure to Evaluate the In Vitro Performance of the Putative Hemangioblastoma Neovascularization Using the Spheroid Sprouting Assay

This paper presents a comprehensive procedure to evaluate in vitro whether classic tumor angiogenesis exists in hemangioblastomas (HBs) and its role in HBs. The results highlight the complexity of HB-neovascularization and suggest that this common form of angiogenesis is only a complementary mechanism in the HB-neovascularization.

用球形发芽法评价 Hemangioblastoma 新生血管的体外性能的综合程序

The inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene plays a crucial role in the development of hemangioblastomas (HBs) within the human ...

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful immunotherapies. Several studies have indicated that tumor infiltrating myeloid cells (e.g., myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs)) suppress cytotoxicity of CD8+ T cells in the tumor microenvironment, and that targeting these regulatory myeloid cells can improve immunotherapies. Here, we present an in vitro assay system to evaluate immune suppressive effects of monocytic-MDSCs and TAMs on the tumor-killing ability of CD8+ T cells. To this end, we first cultured na&#239;ve splenic CD8+ T cells with anti-CD3/CD28 activating antibodies in the presence or absence of suppressor cells, and then co-cultured the pre-activated T cells with target cancer cells in the presence of a fluorogenic caspase-3 substrate. Fluorescence from the substrate in cancer cells was detected by real-time fluorescence microscopy as an indicator of T-cell induced tumor cell apoptosis. In this assay, we can successfully detect the increase of tumor cell apoptosis by CD8+ T cells and its suppression by pre-culture with TAMs or MDSCs. This functional assay is useful for investigating CD8+ T cell suppression mechanisms by regulatory myeloid cells and identifying druggable targets to overcome it via high throughput screening.

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

We describe here a protocol to investigate cytotoxicity of pre-activated CD8+ T cells against cancer cells by detecting apoptotic cancer cells via real-time microscopy. This protocol can investigate mechanisms behind myeloid cell-induced T cell suppression and evaluate compounds aimed at replenishing T cells via blockade of immune suppressive myeloid cells.

cd8+ t 细胞诱导体外肿瘤细胞凋亡的实时检测--研究肿瘤浸润髓内细胞的免疫抑制作用

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful ...

Droplet Digital TRAP (ddTRAP): Adaptation of the Telomere Repeat Amplification Protocol to Droplet Digital Polymerase Chain Reaction

The telomere repeat amplification protocol (TRAP) is the most widely used assay to detect telomerase activity within a given a sample. The polymerase chain reaction (PCR)-based method allows for robust measurements of enzyme activity from most cell lysates. The gel-based TRAP with fluorescently labeled primers limits sample throughput, and the ability to detect differences in samples is restricted to two fold or greater changes in enzyme activity. The droplet digital TRAP, ddTRAP, is a highly sensitive approach that has been modified from the traditional TRAP assay, enabling the user to perform a robust analysis on 96 samples per run and obtain absolute quantification of the DNA (telomerase extension products) input within each PCR. Therefore, the newly developed ddTRAP assay overcomes the limitations of the traditional gel-based TRAP assay and provides a more efficient, accurate, and quantitative approach to measuring telomerase activity within laboratory and clinical settings.

Droplet Digital TRAP ddTRAP: Adaptation of the Telomere Repeat Amplification Protocol to Droplet Digital Polymerase Chain Reaction

We successfully converted the standard telomere repeat amplification protocol (TRAP) assay to be employed in droplet digital polymerase chain reactions. This new assay, called ddTRAP, is more sensitive and quantitative, allowing for better detection and statistical analysis of telomerase activity within various human cells.

液滴数字 TRAP (ddTRAP): 对端粒重复放大协议的适应性, 以实现液滴数字聚合酶链反应

The telomere repeat amplification protocol (TRAP) is the most widely used assay to detect telomerase activity within a given a sample. The polymerase ...

Optimization, Design and Avoiding Pitfalls in Manual Multiplex Fluorescent Immunohistochemistry

Microenvironment evaluation of intact tissue for analysis of cell infiltration and spatial organization are essential in understanding the complexity of disease processes. The principle techniques used in the past include immunohistochemistry (IHC) and immunofluorescence (IF) which enable visualization of cells as a snapshot in time using between 1 and 4 markers. Both techniques have shortcomings including difficulty staining poorly antigenic targets and limitations related to cross-species reactivity. IHC is reliable and reproducible, but the nature of the chemistry and reliance on the visible light spectrum allows for only a few markers to be used and makes co-localization challenging. Use of IF broadens potential markers but typically relies on frozen tissue due to the extensive tissue autofluorescence following formalin fixation. Flow cytometry, a technique that enables simultaneous labeling of multiple epitopes, abrogates many of the deficiencies of IF and IHC, however, the need to examine cells as a single cell suspension loses the spatial context of cells discarding important biologic relationships. Multiplex fluorescent immunohistochemistry (mfIHC) bridges these technologies allowing for multi-epitope cellular phenotyping in formalin fixed paraffin embedded (FFPE) tissue while preserving the overall microenvironment architecture and spatial relationship of cells within intact undisrupted tissue. High fluorescent intensity fluorophores that covalently bond to the tissue epitope enables multiple applications of primary antibodies without worry of species specific cross-reactivity by secondary antibodies. Although this technology has been proven to produce reliable and accurate images for the study of disease, the process of creating a useful mfIHC staining strategy can be time consuming and exacting due to extensive optimization and design. In order to make robust images that represent accurate cellular interactions in-situ and to mitigate the optimization period for manual analysis, presented here are methods for slide preparation, optimizing antibodies, multiplex design as well as errors commonly encountered during the staining process.

Multiplex fluorescent immunohistochemistry is an emerging technology that enables the visualization of multiple cell types within intact formalin-fixed, paraffin embedded (FFPE) tissue. Presented are guidelines for ensuring a successful 7-color multiplex with instructions for optimizing antibodies and reagents, preparing slides, design and tips for avoiding common problems.

手动复用荧光免疫学的优化、设计和避免陷阱

Microenvironment evaluation of intact tissue for analysis of cell infiltration and spatial organization are essential in understanding the complexity of ...

ATAC-Seq Optimization for Cancer Epigenetics Research

The assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) probes deoxyribonucleic acid (DNA) accessibility using the hyperactive Tn5 transposase. Tn5 cuts and ligates adapters for high-throughput sequencing within accessible chromatin regions. In eukaryotic cells, genomic DNA is packaged into chromatin, a complex of DNA, histones, and other proteins, which acts as a physical barrier to the transcriptional machinery. In response to extrinsic signals, transcription factors recruit chromatin remodeling complexes to enable access to the transcriptional machinery for gene activation. Therefore, identifying open chromatin regions is useful when monitoring enhancer and gene promoter activities during biological events such as cancer progression. Since this protocol is easy to use and has a low cell input requirement, ATAC-seq has been widely adopted to define open chromatin regions in various cell types, including cancer cells. For successful data acquisition, several parameters need to be considered when preparing ATAC-seq libraries. Among them, the choice of cell lysis buffer, the titration of the Tn5 enzyme, and the starting volume of cells are crucial for ATAC-seq library preparation in cancer cells. Optimization is essential for generating high-quality data. Here, we provide a detailed description of the ATAC-seq optimization methods for epithelial cell types.

ATAC-seq is a DNA sequencing method that uses the hyperactive mutant transposase, Tn5, to map changes in chromatin accessibility mediated by transcription factors. ATAC-seq enables the discovery of the molecular mechanisms underlying phenotypic alterations in cancer cells. This protocol outlines optimization procedures for ATAC-seq in epithelial cell types, including cancer cells.

用于癌症表观遗传学研究的ATAC-Seq优化

The assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) probes deoxyribonucleic acid (DNA) accessibility using the ...

通过量化单细胞力学增强靶向癌症诊断

Shear Assay Protocol for the Determination of Single-Cell Material Properties

Irregular biomechanics are a hallmark of cancer biology subject to extensive study. The mechanical properties of a cell are similar to those of a material. A cell's resistance to stress and strain, its relaxation time, and its elasticity are all properties that can be derived and compared to other types of cells. Quantifying the mechanical properties of cancerous (malignant) versus normal (non-malignant) cells allows researchers to further uncover the biophysical fundamentals of this disease. While the mechanical properties of cancer cells are known to consistently differ from the mechanical properties of normal cells, a standard experimental procedure to deduce these properties from cells in culture is lacking. 
This paper outlines a procedure to quantify the mechanical properties of single cells in vitro using a fluid shear assay. The principle behind this assay involves applying fluid shear stress onto a single cell and optically monitoring the resulting cellular deformation over time. Cell mechanical properties are subsequently characterized using digital image correlation (DIC) analysis and fitting an appropriate viscoelastic model to the experimental data generated from the DIC analysis. Overall, the protocol outlined here aims to provide a more effective and targeted method for the diagnosis of difficult-to-treat cancers.

作者聚焦：用于测定单细胞材料特性的剪切测定实验方案  

This protocol outlines the quantification of the mechanical properties of cancerous and non-cancerous cell lines in vitro. Conserved differences in the mechanics of cancerous and normal cells can act as a biomarker that may have implications in prognosis and diagnosis.