Name: an automated differential nuclear staining assay for accurate determination of mitocan cytotoxicity
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NVK，癌症研究的CPRIT学者，感谢德克萨斯癌症预防和研究所（CPRIT）的慷慨支持，CPRIT授予RR150044。这项工作还得到了韦尔奇基金会研究赠款C-1930和国家卫生研究院R35 GM129294授予NVK的支持。资助者在研究设计、数据收集和分析、决定出版或准备手稿方面没有作用。

1. 细胞毒性测定：设置
<ol><li>在适当的介质（无血清或 1、2.5 或 5% FBS RPMI-1640）中，以所需浓度准备感兴趣的化合物溶液。<ol>
<li>要测量单个化合物的细胞毒性（例如，确定有效剂量），以2倍的最终浓度准备化合物。</li>
		<li>要测量化合物组合的细胞毒性，以4倍的最终浓度准备化合物。</li>
		<li>通过将相同数量的溶剂与适当的介质混合，准备仅溶剂控制。例如，如果测试化合物溶解在DMSO和甲醇...

<ol>
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尽管 Hoechst/PI 细胞毒性测定的协议是可靠的，需要相对少的动手时间，但有几个实验细节对于确保准确结果非常重要。首先，必须确保DMSO浓度低于0.5%（v/v）。人们普遍认为，即使低剂量的DMSO暴露也可以显著改变细胞的形态和附着，并显著延迟细胞周期进展39，40。
第二，在治疗后应尽快进行染色。由于没有洗涤步骤，化合物留在?...

确定有效癌症治疗的第一步是选择一种稳健、无偏的细胞毒性检测，可用于检查治疗的效果。低通量实验的一个常见选择是将锥蓝染料排除在活细胞之外。这种方法之所以受到青睐，是因为它允许采用相对公正的方法量化细胞存活率。trypan蓝色被动地扩散到膜受损的细胞中，但它被有效地阻止进入健康的细胞1。活细胞和总细胞的商数代表生存能力的百分比，这表示治疗的功效。trypan 蓝色测定最大的缺点是它不适合高通量方法。它具有相对较低的信噪比，由于活细胞的染色，长时间的染色可能导致伪影。因此，trypan 蓝色排除通常是，但并不总是2，降级到手动计数。这使得它太慢，并引入了由于研究人员的主观判断（除非使用致盲或独立计数，这进一步降低实验室的吞吐量）的强烈偏见的可能性）。一般来说，这种测定的通量不足以发现现代药物。
可行性测定通常具有更高的吞吐量，使研究人员能够绕过这一限制，但需要注意一些重大警告（参见表1）。这些方法通常分为两组。一组由基于细胞氧化酶功能的色度测定组成。无色或非荧光基板可转换为充满活力的产品，可使用分光光度计进行量化。经典示例包括四分之一盐（MTT、WST-1、XTT 等）和雷萨祖林。此类别还包括利用荧光素评估 ATP 水平的发光检测。这种类型的分析有其潜在的限制，即它们正在测量细胞代谢，这本身不是细胞生存能力。细胞在不利条件下变得静止是很常见的，但仍保留分裂3、4、5的能力。例如，癌症干细胞通常相对代谢静默6，7，8，9，并且很可能很难使用这些技术进行检测。损害线粒体功能的治疗的有效性，如大多数米托坎，也可能被大大高估。
另一种方法利用各种物质的化学性质，允许它们交叉或不交叉生物膜。一个例子是核污渍，如SYTOX或碘化二甲（PI）。此类别还包括在概念上相似但功能不同的测定，如乳酸脱氢酶 （LDH） 测定，它测量 LDH 释放到细胞外环境作为细胞坏死的指标（图1，表 1）。这些测定更能够区分代谢不活跃细胞和死细胞。
<table border="1" fo:keep-together.within-page="1"><tbody>
<tr>
<td>检测/染色</td>
			<td>检测到的细胞死亡类型</td>
			<td>必要设备</td>
			<td>主要功能</td>
		</tr>
<tr>
<td>MTT， CKK-8， 阿拉玛蓝 （雷萨祖林）</td>
			<td>凋亡/坏死</td>
			<td>分 光 光度计</td>
			<td>价格便宜，速度快;端点测定;依赖于酶的活性（在MTT的情况下完全线粒体），并且不区分细胞死亡模式1，10
</td>
		</tr>
<tr>
<td>LDH 版本</td>
			<td>坏死</td>
			<td>分 光 光度计</td>
			<td>快速，独立于线粒体酶的活性;高通量测试费用昂贵;检测坏死细胞与复合血浆膜11，12
</td>
		</tr>
<tr>
<td>试盘蓝色 （TB）</td>
			<td>凋亡/坏死</td>
			<td>显微镜</td>
			<td>细胞不渗透剂;不区分细胞死亡模式;劳动，不适合高通量筛选;更难与粘附细胞一起使用;容易对用户的主观判断，但被认为是标准细胞生存能力测量方法13
</td>
		</tr>
<tr>
<td rowspan="2">阿克里丁橙 （AO）</td>
			<td>凋亡/坏死/</td>
			<td rowspan="2">荧光显微镜</td>
			<td rowspan="2">具有独特光谱特性的核酸染料，可区分凋亡和坏死/坏死14
</td>
		</tr>
<tr>
<td>坏死</td>
		</tr>
<tr>
<td>霍赫斯特 33342， 达皮</td>
			<td>凋 亡</td>
			<td>荧光显微镜或流动环流仪</td>
			<td>细胞渗透;不宜自己监测细胞死亡;有用的共同染色;可用于评估早期凋亡中的染色质凝结和核分裂;可与碘化丙二基配对，以区分凋亡与坏死15，16
</td>
		</tr>
<tr>
<td>碘化（PI）</td>
			<td>晚期凋亡/坏死</td>
			<td>荧光显微镜或流动环流仪</td>
			<td>细胞不渗透的干扰器;检测细胞死亡的晚期凋亡和坏死模式17。长期孵育后有毒和可渗透18
</td>
		</tr>
</tbody></table>表1.细胞毒性测定列表。细胞毒性测定，其中一些用于本研究，列及其主要特征的简要描述。
最近的研究表明，线粒体代谢在一些癌症中改变19，20，21，22，23，24，25。例如，急性骨髓性白血病（AML）已被证明可调节其线粒体质量、mtDNA含量和线粒体呼吸，以满足其能量需求19、26、27。另一方面，一些实体肿瘤的特点是线粒体功能障碍，或者更确切地说是"代谢重新编程"，如对涉及OXPHOS的线粒体蛋白的降低调节或mtDNA含量降低，这一点与肿瘤侵入性、转移性潜力和抗凋亡诱导药物28、29有关。此外，最近，人们对于使用影响线粒体功能的机械多样化化合物（通常称为米托坎30）作为特定癌症的潜在疗法的兴趣也日益增加。这些药物针对ATP合成，线粒体DNA，OXPHOS和ROS生产，以及亲凋亡和抗凋亡蛋白相关的线粒体30，31。多项研究表明，这种方法有显著的希望19，32，33，34。然而，这些代谢偏差在癌细胞生物学或线粒体靶向治疗可能会显著影响传统的生存能力测定，基于线粒体功能。
这里介绍了差分核染色测定的优化方案。该协议允许快速和准确地测定米托坎的细胞毒性或它们与其他化合物的组合。Hoechst 33342 是一种细胞渗透性核染料，它很容易穿过细胞膜来染色 DNA，从而获得细胞总数。通过与只进入死细胞核的PI共同染色，可以准确确定活细胞（仅霍赫斯特）和死细胞（两者染色）的比例。该协议通过添加一个步骤来优化染料浓度（通过用正交 trypan 蓝色方法交叉引用结果）和在成像前离心板来优化已发布的测定35。 由于许多细胞系是半粘附的或悬浮的，离心会增加图像化的细胞比例，并显著提高精度。该检测有几个优点，包括染色不需要去除介质或洗涤。染料混合物价格低廉，易于准备，并且与多通道/机器人移液系统兼容。
细胞被染色后，用自动显微镜成像。这还有一个好处，即创建图像的永久记录，以后可以重新分析，并且可以通过对捕获的图像进行目视检查来重新评估特定化合物的效果。获得图像后，可以手动或使用多个软件包中的任何一个进行计数，包括免费（例如，ImageJ、CellProfiler 等）和商业软件（例如，变形、Gen5 等）。自动细胞计数通常更可取，因为正确开发的自动细胞计数管道比手动计数更准确，偏置更少。它们还更有效地忽略细胞碎片或不溶性复合物。这些管道的开发通常很简单，并且被使用的污渍的效率简化。输出是定量的，因为死细胞的实际数量是自动计算的总细胞数，并且可以应用不同的阈值来增加或减少检测35 的串数。为方便起见，包括使用 Gen5 v. 3.00 软件兼容 Cytation 5 细胞成像多模式读取器计算细胞的优化参数。

<table border="0" cellpadding="0" cellspacing="0" style="width:1423px;" width="1423">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">2-Deoxy-D-glucose/2-DG</td>
			<td style="width:137px;">Chem-Impex</td>
			<td style="width:176px;">50-519-067</td>
			<td style="width:819px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">3-bromo-pyruvate</td>
			<td>Alfa Aesar</td>
			<td style="width:176px;">1113-59-3</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">96-Well plates</td>
			<td style="width:137px;">Greiner Bio-One</td>
			<td style="width:176px;">655090</td>
			<td>Black or clear flat-bottomed 96-well plates 
			</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alamar blue HS cell viability reagent (100mL)&#160;</td>
			<td style="width:137px;">Thermo Fisher</td>
			<td>A50101</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Countess II automated cell counter</td>
			<td style="width:137px;">Thermo Fisher</td>
			<td style="width:176px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cytation 5 Cell Imaging Multi-Mode Reader</td>
			<td style="width:137px;">BioTek</td>
			<td style="width:176px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Hoechst 33342</td>
			<td style="width:137px;">Thermo Fisher</td>
			<td style="width:176px;">62249</td>
			<td>20 mM solution; final concentration 1:1,000 
			</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HyClone fetal bovine serum</td>
			<td>GE Healthcare</td>
			<td>#25-514</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">m-chlorophenylhydrazone/CCCP</td>
			<td>Sigma Aldrich</td>
			<td style="width:176px;">C2759</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">PBS tablets</td>
			<td style="width:137px;">Thermo Fisher</td>
			<td>BP2944100</td>
			<td>1 tablet + 200 mL of sterile water = 1x PBS solution 
			</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin-Streptomycin-Glutamine (100X)</td>
			<td>Gibco</td>
			<td>10378016</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Pierce LDH assay kit</td>
			<td style="width:137px;">Thermo Fisher</td>
			<td>50-103-5952</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Propidium Iodide</td>
			<td style="width:137px;">Thermo Fisher</td>
			<td>50-596-072</td>
			<td>Dry powder; stock 1 mg/mL in PBS; final concentration 5 &#181;g/mL (leukemia cells), 1 &#181;g/mL (normal PBMCs) 
			</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Rotenone</td>
			<td>Ark Pharm</td>
			<td style="width:176px;">AK115691</td>
			<td></td>
		</tr>
		<tr height="83">
			<td height="83" style="height:84px;width:291px;">RPMI-1640 Medium 
			With L-glutamine and sodium bicarbonate, liquid, sterile-filtered, suitable for cell culture</td>
			<td style="width:137px;">Sigma Aldrich</td>
			<td style="width:176px;">R8758-500ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Thiazolyl blue tetrazolium bromide</td>
			<td style="width:137px;">ACROS Organics</td>
			<td style="width:176px;">AC158990010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Trypan blue stain (0.4%)</td>
			<td style="width:137px;">Gibco</td>
			<td style="width:176px;">15250-061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;width:1423px;">Cell lines</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">K562</td>
			<td style="width:137px;">ATCC</td>
			<td style="width:176px;">CCL-243</td>
			<td>CML cell line 
			</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">MOLM-13</td>
			<td style="width:137px;">ATCC</td>
			<td style="width:176px;"></td>
			<td>AML cell line 
			</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">MOLT-4</td>
			<td style="width:137px;">ATCC</td>
			<td>CRL-1582</td>
			<td>ALL cell line 
			</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">OCI-AML2</td>
			<td style="width:137px;">ATCC</td>
			<td style="width:176px;"></td>
			<td>AML cell line 
			</td>
		</tr>
	</tbody>
</table>

an automated differential nuclear staining assay for accurate determination of mitocan cytotoxicity

线粒体对致癌转化的贡献是一个广泛关注和积极研究的课题。随着癌症代谢领域变得越来越复杂，使用各种化合物（所谓的米托坎）瞄准线粒体的目标正变得相当流行。不幸的是，许多现有的细胞毒性测定，如那些基于四佐盐或雷萨祖林需要功能线粒体酶，以他们的表现。以线粒体为目标的化合物造成的损害往往会损害这些测定的准确性。在这里，我们描述了一个基于两种荧光染料的差分染色的修饰协议，其中一种是细胞渗透剂（Hoechst 33342），另一种不是（碘化二氧化二钠）。染色的差异允许活细胞和死细胞被区分。该检测可支持自动显微镜和图像分析，可提高吞吐量并减少偏差。这也允许使用96孔板以高通量的方式使用测定，这使得它成为药物发现努力的可行选择，特别是当有关药物具有一定水平的米托毒性时。重要的是，Hoechst/PI染色测定获得的结果表明，在测定与其他方法进行比较时，与尝试蓝色排除结果和生物复制之间，一致性都有所提高。

上述协议是使用OCI-AML2细胞开发的，该细胞作为具有代表性的急性骨髓性白血病细胞系。AML的特点是骨髓26中未分化和非功能性造血细胞的异常增殖。尽管 AML 靶向治疗最近有所发展，但护理标准几十年来一直保持不变，包括诱导疗法（通常包括三天的环丙氨酸，例如，多诺鲁比辛、伊达鲁比辛或安赛迪翁尼米托桑特龙，以及7天的环丙氨酸），然后是整合（通常包括环丙氨酸治疗...

Watch this Scientific Journal Video about An Automated Differential Nuclear Staining Assay for Accurate Determination of Mitocan Cytotoxicity at JoVE.com

An Automated Differential Nuclear Staining Assay for Accurate Determination of Mitocan Cytotoxicity

该协议描述了一种快速、高通量、可靠、廉价和公正的检测方法，用于有效确定细胞的可行性。当细胞的线粒体受损时，这种测定特别有用，这干扰了其他检测。该测定使用自动计数细胞染色的两种核染料 - Hoechst 33342 和碘化丙二钠。

自动差分核染色测定，准确测定三元细胞毒性

The contribution of mitochondria to oncogenic transformation is a subject of wide interest and active study. As the field of cancer metabolism becomes ...

这种方法将允许研究人员评估细胞生存能力独立于线粒体功能。并且它支持高通量药物筛查工作。该技术快速、准确、廉价，可测定化合物细胞毒性，包括大多数细胞类型的线粒体功能。该协议易于执行。重视药物制备、治疗条件和细胞密度，确保实验的准确性和有效性。此方法的可视化演示非常重要，因为图像采集和图像分析步骤可能难以概括，尤其是对于对 Gen5 软件经验很少的研究人员。首先，在所需浓度下制备感兴趣的化合物溶液。始终确保通过彻底涡旋混合化合物。要测量单个化合物的细胞毒性，以2倍的最终浓度准备化合物。如果测量化合物组合的细胞毒性，以4倍的最终浓度进行准备。仅通过将相同数量的溶剂与适当的介质混合来准备溶剂控制。将细胞收集到15毫升圆锥管和10微升细胞悬浮液中。对于 trypan 蓝色染色，在等分中加入 10 微升 0.4% trypan 蓝色，并使用血细胞计或细胞计数器来计数可行和非活细胞。在15毫升管中，以200次G为单位的颗粒细胞5分钟，然后吸气或去吸上分。我们暂停细胞颗粒在散文适当的介质在细胞密度的三倍10至第五细胞每毫升。使用多通道移液器将50微升的电池悬浮液播种到96井板的每个井中。对于单化合物测定，将50微升2X化合物溶液加入到每一井中。对于溶剂控制井，加入15微升含有溶剂的测试介质，浓度为2倍。对于组合测定，将每个4X化合物的25微升添加到每个井中。对于单化合物控制井，每 4X 复合溶液和测试介质添加 25 微升。确保 DMSO 的最终浓度不超过 0.5%为确保可重复性，请添加带移液器尖端接触井侧的药物。小心把药物加到不同的地方。这种刺应该执行非常快，最好需要不超过30分钟每板。轻轻敲击板，混合井内装物，在37摄氏度和5%的二氧化碳下孵育。当准备用 Hoechst 33342 和碘化丙二的二氧化二烯染色细胞染色时，准备新鲜的 10X 染色溶液，并在每个井中加入 10 微升。轻轻敲拍盘子，在37摄氏度下孵育15分钟。将板在 200 倍 G 下离心 4 分钟，将所有电池带到板的底部。然后用潮湿的实验室擦拭小心地擦拭板的底部，以清除任何可能干扰成像的碎屑。离心后 15 分钟内对板进行成像。打开 Gen5 软件并创建新的标准协议，单击程序并选择要使用的板类型。通常，96个黑色塑料板。接下来，将读取方法设置为图像，单击"确定"，然后选择具有 4 倍放大目标的 DAPI 和德州红色滤镜集。无需偏移，因为井中心将被成像。对于 DAPI 滤波器，将 LED 设置为 10，将集成时间设置为 99，增益为零。对于德州红，将 LED 设置为 8，集成时间设置为 950，增益为 18。单击"选项"，确保自动对焦在 DAPI 通道上执行，并且通道之间的对焦没有偏移。单击"确定"保存协议。现在可以使用"读取新"按钮录制图像。记录图像后，单击"减少数据"并选择图像预处理。使用基于 DAPI 信号的自动拼合大小应用具有深色背景减法的图像预处理。对于德州红，使用与通道 1 相同的选项。完成后，单击"确定"。然后转到蜂窝分析面板，根据转换的 DAPI 信号应用核掩码，阈值为 6，000 个单位，最小对象大小为 5 微米，最大对象大小为 25 微米。分析整个图像，排除图像边框上的主要边缘对象和拆分接触对象。在高级检测选项中，将滚球直径设置为 30 微米，并基于最低像素的 5% 评估背景。仅将单元格计数保留为计算指标。之后，根据平均变换的德州红信号执行亚填充分析，阈值为 5，000 个单位，以计算死细胞。最后，访问生成的单元格计数。此处显示了沾有 Hoechst、碘化钠的细胞的代表性图像。细胞总数相当高，并且大于死细胞的数量。当一组细胞毒性测定与尝试蓝排除进行比较时，发现MTT和阿拉玛蓝测定结果对细胞的生存能力测量不准确。这些线粒体酶为基础的测定显示，与尝试蓝色排除相比，其可行性显著降低。使用 Hoechst 33342 和 PI 的双染色具有鲁棒性、灵敏度和与 trypan 蓝色染色的最佳结合，以及 CCP 或 2-DG 处理后与 trypan 蓝色排除方法的最小中位数偏差。Hoechst PI 文章还有效地确定细胞在治疗后与线粒体目标分子 Rotenone 和三溴基鲁酮的细胞生存能力。它得到了进一步验证与白血病细胞系面板。这些细胞在Rotenone敏感性中变化不一，从非常敏感到抗性细胞。检测性能不当可能会损害其准确性。此处显示了几个受损的结果。过度污染 PI、忽略离心步骤或在 Hoechst 通道中过度曝光将导致效果欠佳。定时此协议时，请记住优化每个细胞系的染料浓度和染色时间。此外，为确保成像质量，样品板的分分化是必要的。在确定细胞毒性对癌细胞有作用的化合物后，研究人员可以进行机械研究，以确定其相关目标。与传统的MTT测定技术并行筛选小分子库，将允许对线粒体功能的分子快速识别。

Research

JoVE Journal

Cancer Research

A GPC3-targeting Bispecific Antibody, GPC3-S-Fab, with Potent Cytotoxicity

GPC3-targeting 双特异性抗体, GPC3-S-Fab, 具有强大的细胞毒性

This protocol describes the construction and functional studies of a bispecific antibody (bsAb), GPC3-S-Fab. bsAbs can recognize two different epitopes ...

科研

癌症研究

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

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

A Simple Migration/Invasion Workflow Using an Automated Live-cell Imager

使用自动活单元成像仪的简单迁移/入侵工作流

Cancer cell mobility is crucial for the initiation of metastasis. Therefore, investigation of the cell movement and invasive capacity is of great ...

A Practical Guide for the Production and PET/CT Imaging of 68Ga-DOTATATE for Neuroendocrine Tumors in Daily Clinical Practice

A Practical Guide for the Production and PET/CT Imaging of 68Ga-DOTATATE for Neuroendocrine Tumors in Daily Clinical Practice

68型 Ga-DOTATATE 神经内分泌肿瘤的日常临床应用指南

Neuroendocrine tumors are a rare form of cancer that arise from neuroendocrine cells and can be present at almost any location throughout the body. ...

Spatial and Temporal Control of Murine Melanoma Initiation from Mutant Melanocyte Stem Cells

突变黑色素细胞干细胞引发小鼠黑色素瘤的时空控制

Cutaneous melanoma is well known as the most aggressive skin cancer. Although the risk factors and major genetic alterations continue to be documented ...

In Vitro Assay to Study Tumor-macrophage Interaction

体外测定研究肿瘤-巨噬菌体相互作用

Tumor associated macrophages (TAMs) account for a large percentage of cells in the tumor mass for different types of cancers. Glioblastoma (GBM), a ...

Management of Respiratory Motion Artefacts in 18F-fluorodeoxyglucose Positron Emission Tomography using an Amplitude-Based Optimal Respiratory Gating Algorithm

Management of Respiratory Motion Artefacts in 18F-fluorodeoxyglucose Positron Emission Tomography using an Amplitude-Based Optimal Respiratory Gating Algorithm

使用基于振幅的最佳呼吸门控算法对 18个F-氟二氧氧葡萄糖正电子发射断层扫描中的呼吸运动人工制品进行管理

Positron emission tomography (PET) combined with X-ray computed tomography (CT) is an important molecular imaging platform that is required for accurate ...

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

正交胰腺肿瘤的产生与肿瘤渗透T细胞细胞毒性的外体特征

In vivo models of pancreatic cancer provide invaluable tools for studying disease dynamics, immune infiltration and new therapeutic strategies. The ...

Immunofluorescence Imaging of DNA Damage and Repair Foci in Human Colon Cancer Cells

人类结肠癌细胞DNA损伤与修复福西的免疫荧光成像

The purpose of the manuscript is to provide a step-by-step protocol for performing immunofluorescence microscopy to study the radiation-induced DNA damage ...

Three Differential Expression Analysis Methods for RNA Sequencing: limma, EdgeR, DESeq2

RNA 测序的三种微分表达分析方法：利马、边缘、DESeq2

RNA sequencing (RNA-seq) is one of the most widely used technologies in transcriptomics as it can reveal the relationship between the genetic alteration ...