我们感谢 Mrs. 子柴田的技术援助。这项工作得到了来自日本教育、文化、体育、科学和技术部的补助金的支持, 这些项目是为领导研究生院提供的, 培养了在重离子治疗和工程方面的全球领导者。

1. 材料的制备 <ol> <li> 高压釜盖板。
	<ol> <li> 将盖子卡瓦放在玻璃烧杯中. </li>
		<li> 用铝箔盖住玻璃烧杯. </li>
		<li> 121 和 #176 高压釜; C at 15 psi, 20 分钟. </li>
		<li> 50 和 #176 干燥; c. 在室温下存放在一个文化罩里. </li>
	</ol> </li> <li> 准备固定解决方案: 3% 甲醛 + 2% 蔗糖在磷酸盐缓冲盐水 (PBS)。
	<ol> <li> 到1000毫升玻璃瓶, 添加700毫升 PBS 和30克甲醛. 
		注意: 甲醛有腐蚀性, 佩戴合适的手套. </li>
		<li> 通过加热到80和 #176 完全溶解甲醛; C 使用微波. 
		注意: 甲醛烟雾有毒, 戴上口罩. </li>
		<li> 在室温下过夜. </li>
		<li> 添加20克蔗糖. </li>
		<li> 添加 PBS 以使总卷1升. </li>
		<li> 分50毫升管。固定液可储存3年, 在-20 和 #176; c 或3月, 在4和 #176; c. </li>
	</ol> </li> </ol> 2。细胞培养的制备 注意: 这些过程应在文化引擎罩中执行. 
<ol> <li> 培养和通道 U2OS 人骨肉瘤细胞维持对数增长 5 。
	注: 在确定最佳辐照剂量后可使用其他细胞类型. </li>
	<li> 用70% 乙醇浸湿的纸巾擦拭手术刀。使用手术刀, 在35毫米细胞培养皿上放置一个盖子滑动 (以下简称和 #34; 菜和 #34;). 
	注: 根据实验设计, 一盘中的盖板数量可以增加 (见讨论). </li>
	<li> 添加 1 mL 培养基: DMEM 补充10% 胎牛血清. 
	注意: 其他介质可根据所选的单元格类型使用. </li>
	<li> 用70% 乙醇浸湿的纸巾擦拭镊子。使用镊子, 轻轻地握住盖子滑动的中心。完全从盘中吸取培养基, 同时保持盖子滑动. 
	#8203; 注意: 这一步促进了固定的盖子滑到盘子. </li>
	<li> 使用胰蛋白酶 5 分离粘附培养的单元格。
	<ol> <li> 从盘中吸取文化介质. </li>
		<li> 添加1毫升 PBS, 轻轻摇动盘子. </li>
		<li> 从盘中吸出 PBS. </li>
		<li> 添加1毫升胰蛋白酶 [0.25w/v% Trypsin-1mmol/L EDTA] 到该菜. </li>
		<li> 孵育5分钟, 在37和 #176; C 在包含 5% CO 2 的大气层中. </li>
		<li> 将9毫升培养基添加到盘中. </li>
		<li> 使用1000和 #956; L 微, 准备一个单细胞悬浮液. </li>
	</ol> </li> <li> 计数单元格 5 。
	<ol> <li> 在1.5 毫升管中, 添加0.9 毫升细胞悬浮液和0.1 毫升0.4% 台盼蓝溶液. </li>
		<li> 应用10和 #956; L 到例. </li>
		<li> 在倒置相显微镜下检查不 ( 即 活) 细胞的数量. </li>
	</ol> </li> <li> 在15毫升的管中, 在培养基中制备3毫升细胞悬浮液, 密度为 0.5 x 10 5 细胞/毫升。
	注意: 可以根据实验需要修改单元格密度 (请参见 讨论 ). </li>
	<li> 轻轻地将电池悬浮液的2毫升加入到盘中. </li>
	<li> 在37和 #176 的夜间孵育; C 在包含 5% CO 2 的大气层中. </li>
</ol> 3。辐照 注意: 根据制造商和 #39 的说明, 仔细处理 X 射线器. 
<ol> <li> 在倒置相显微镜下检查单元格。确认这些单元格已连接到盖板滑和活. </li>
	<li> 用 6 X 射线照射盘子 (1.4 个/分钟, 300 KVP, 20 毫安). </li>
	<li> 在37和 #176 中孵育72小时; C 在包含 5% CO 2 的大气层中进行。
	注意: 可以根据实验设计修改孵化时间 (参见 讨论 ). </li>
</ol> 4。固定 <ol> <li> 从盘中吸取培养基. </li>
	<li> 使用剪刀, 切1000和 #956 的尖端; 从最后微小费5毫米. 
	注: 切口尖端有助于顺利应用固定液. </li>
	<li> 使用切口尖端, 将1毫升固定液 (在步骤1.2 中准备) 添加到盘子侧面壁的盘中. 
	注意: 此步骤具有时效性, 因此在处理多个示例时不更改微提示。将固定液直接应用于碟底, 会损伤细胞. </li>
	<li> 将菜放在方形培养皿中。轻轻摇动方形培养皿, 均匀地分布在盖子滑动上的固定液. </li>
	<li> 室温孵育10分钟. </li>
	<li> 从盘中抽出固定液. </li>
	<li> 在盘子的侧壁上加入2毫升 PBS. 
	注意: PBS 直接在盘子底部的应用会损坏细胞. </li>
	<li> 从盘中吸出 PBS. </li>
	<li> 重复步骤4.7 和4.8 两次. 
	注: 该盘可存放于4和 #176 的1周; C 与2毫升 PBS 沐浴的盖子滑动. </li>
</ol> 5。DAPI 染色 <ol> <li> 到幻灯片玻璃, 应用5和 #956; L 1 和 #956; g/毫升 DAPI 染色试剂. 
	注: DAPI 染色试剂6月后稳定保存, 不受光照或低于-20 #176; C. </li>
	<li> 使用手术刀, 取下盘中的盖子滑. </li>
	<li> 通过用纸巾接触盖子滑块的边缘, 在盖板上画出多余的 PBS. </li>
	<li> 将盖子滑动倒置到滑动玻璃上的 DAPI 染色试剂滴上, 使细胞暴露在 DAPI 染色试剂中. 
	注意: 此步骤具有时效性。干燥的 DAPI 染色试剂可导致次优染色. </li>
	<li> 该示例可在-20 和 #176 中存储1年; C. </li>
</ol> 6。图像获取 <ol> <li> 在荧光显微镜上检查样品。原子核的可视化使用 DAPI 过滤器. 
	注: 根据研究员和 #39 的兴趣, 可以使用20X 或60X 的油透镜。使用60X 油透镜时, 在盖板上加一滴油。要注意样品不要碰到镜片;否则, 镜片可能会损坏. </li>
	<li> 使用 CCD 摄像机和数字图像采集软件获取原子核的图像, 其设置和参数如下: DAPI 滤镜、单层图像、增益1X 和自动曝光. </li>
	<li> 完成图像采集: 用纸巾擦干净的纸巾擦拭20X 镜片。用用氯仿浸湿的纸巾擦拭60X 油透镜. </li>
</ol> 7。克隆细胞死亡模式的评价 <ol> <li> 在随机选取的图像中, 计算符合细胞凋亡、有丝分裂突变和细胞衰老标准的细胞核数, 直到计数的细胞核总数达到300。为每个实验设置执行此步骤. 
	<ol> <li> 细胞凋亡的标准: 存在凋亡体 ( 即 , 凝聚和碎裂的核) 6 , 7 . </li>
		<li> 有丝分裂突变的标准: 显示两个或多个不同的裂片或 micronucl 的细胞核的存在ei 8 , 9 , 10 . </li>
		<li> 细胞衰老的标准: 衰老相关的异病灶 (SAHF) ( 即 , 包含30-50 明亮、致密病灶的细胞核 DNA) 2 , 11 . </li>
	</ol>
	</li>
</ol>

<ol>
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作者声明他们没有竞争的金融利益。

协议中的关键步骤如下。首先, 细胞应作为单层培养皿, 因为对 DAPI 染色的细胞核进行形态学评估对多层细胞是困难的。为此, 在步骤2.9 中, 建议将培养皿小心地转移到孵化器;文化盘的晃动产生悬浮细胞的漩涡, 导致细胞集中在培养皿的中心。此外, overconfluence 导致多层细胞应避免。为此, 在步骤2.7 中, 在培养皿上播种的细胞数量可以根据种群加倍时间和照射与固定的间隔进行修正。建议在固定时汇合约80。其次, 速度是重要的固定和 DAPI 染色 (即, 步骤4和 5)。对于这些步骤所用的时间, 样本间的不一致会导致原子核中 DAPI 信号强度的异质性, 这将使形态学评估模糊不清。
在步骤2.2 中, 单个培养皿中的盖板数量可以增加;最多可以将四套卡瓦放在 35 mm 的盘子里, 用更大的盘子可以进一步增加这个数字。在每个培养皿中放置多个盖板, 可以对给定的处理进行时间课程评估的有效操作 (即, 在多个时间点, 可从文化盘中一个一个地收集封面单)。
在步骤3.2 中, 辐照剂量可以根据研究者的兴趣进行修正。应用一致剂量的多细胞线, 使比较的敏感性, 每个模式的克隆细胞死亡之间的细胞系。另一方面, 使用 iso 克隆生存剂量的每一个细胞线可以比较克隆细胞死亡剖面之间的细胞线。克隆生存剂量可由克隆生存率测定12确定。D10值, 提供10% 克隆生存的剂量, 是 iso 克隆生存剂量的一个常见端点。
3.3 步, 从照射到固定的时间可以根据研究者的兴趣进行修改;这一点很重要, 因为 IR 诱导的细胞凋亡、有丝分裂突变和细胞衰老的峰值时间根据细胞系和治疗的不同而变化。在本文中, 我们使用72小时后辐照作为时间点, 这将是最有用的初始筛选克隆细胞死亡剖面, 基于多项研究, 我们的小组和其他描述如下1,2,3,4,8,9: (i) x 射线诱导的实体肿瘤细胞凋亡主要发生在照射后几天。(ii) x 射线诱发的有丝分裂突变在肿瘤细胞中最显著的发生在第二次或第三次有丝分裂后, 从短暂的细胞周期的释放引起的照射。释放通常发生在大约24小时在辐照以后, 其次由重复的分裂在大约 24 h 的间隔时间. (iii) ray–induced 细胞衰老在一个间隔依赖于问题的细胞系后变得明显: 2 天后辐射为一些早期的情况下, 7 天后, 大多数细胞线照射。在从最初的筛查中获得了克隆细胞死亡剖面的总体图像后, 时间课程实验将提供一个更详细的说明每种模式的克隆细胞死亡在特定的细胞线和/或条件的峰值时间兴趣4。
应该注意的是, DAPI 染色法和克隆生存法, 一个黄金标准的方法, 辐射敏感性评估, 是不可互换的。细胞凋亡和有丝分裂的灾难只持续数小时。因此, DAPI 染色测定的时间点检测细胞凋亡和有丝分裂突变发生在时间点的评估。另一方面, 在特定时间点的克隆生存试验的结果包括在照射后 10-14 天的潜伏期内发生的细胞凋亡和有丝分裂突变的总数量。与细胞凋亡和有丝分裂突变不同, 衰老细胞保留在培养皿上;它们随着时间的推移逐渐累积。因此, DAPI 染色法和克隆生存法的结果都反映了潜伏期内的衰老总量。重要的是, 细胞凋亡的比例、有丝分裂突变和辐照后的衰老, 都有不同程度的差异。从理论上讲, 一项化验的结果不能直接转化为其他化验。
DAPI 染色法有几个局限性。首先, 有丝分裂突变是否是一种独特的细胞死亡模式仍然存在争议。在辐射生物学领域, 有丝分裂突变被认为是一种主要的 IR 诱导细胞死亡模式, 有别于其他机制的克隆细胞死亡1。另一方面, 其他人认为有丝分裂突变不是细胞死亡的一种独特的模式, 而是在细胞死亡之前的过程, 包括细胞凋亡和坏死13,14。因此, 细胞凋亡和有丝分裂突变可能在一定程度上重叠。其次, 先前的研究表明, 细胞衰老可能发生在一些细胞系和治疗设置2SAHF 缺乏。目前, 为每个克隆细胞死亡模式专门设计的其他方法应用于提高某一实验结论的鲁棒性。第三, DAPI 染色法不能评估克隆细胞死亡的模式, 除了细胞凋亡, 有丝分裂突变, 细胞衰老 (如, 坏死和自噬)。第四, 临床上未阐明 DAPI 染色法作为肿瘤放疗疗效预测因子的效用。从这个角度来看, 克隆生存试验, 评估克隆细胞死亡的总金额, 优于 DAPI 染色法, 因为建立了一个相关的 SF2, 细胞存活的分数辐照2X 光和肿瘤对放疗的反应15。然而, 值得注意的是, 克隆存活率测定在临床上没有得到广泛的应用, 主要是因为需要高度的专业知识和很长的时间 (即, 14 天) 来获取数据。相比之下, DAPI 染色法的过程比较简单, 而且耗时较短, 大约 3-4 天, 以产生结果。DAPI 染色的效用检测作为肿瘤对放疗的反应的预测因子, 将在近期临床上进行试验。
总之, DAPI 染色法是一种 cost-effective one-step 测定, 同时评估三主要模式的 IR 诱导克隆细胞死亡。这种方法可以很容易地筛克隆细胞死亡模式的各种细胞线, 治疗设置和时间点, 目的是阐明细胞死亡机制的目标细胞和条件的兴趣。

电离辐射 (IR) 诱导多种模式的克隆细胞死亡。红外诱导克隆细胞死亡的研究对于了解 ir 对正常组织的毒性以及发展提高肿瘤放疗治疗效果的方法具有重要意义。凋亡、有丝分裂突变和细胞衰老是 IR1引起的克隆细胞死亡的主要模式。凋亡是由 DNA 损伤引发的细胞死亡的调控模式1。有丝分裂突变是细胞死亡, 发生由于异常有丝分裂导致的未 DNA 双断裂1。细胞衰老被定义为不可逆细胞生长的状态, 逮捕2;注意细胞衰老不是细胞死亡本身, 而是一种克隆细胞死亡的模式, 因为它能消灭衰老细胞的克隆生存。
各种细胞的检测已经发展到单独评估凋亡, 有丝分裂突变, 和细胞衰老。细胞凋亡可由终端 deoxynucleotidyl 转移酶 dUTP 尼克结束标记 (TUNEL) 染色, 蛋白 V 染色, DNA 碎片测定, sub-G1 细胞周期的流式细胞仪检测1。有丝分裂突变可以通过免疫荧光染色的有丝分裂标记, 包括 MPM2, TUNEL 染色, 和电子显微镜1。细胞衰老可以通过衰老相关的β-乳糖 (SA β-加仑) 染色、生长抑制法和电子显微镜来评估1。重要的是, 克隆细胞死亡的主要模式不同的细胞系和治疗设置3,4。因此, 为了阐明某一特定实验环境下克隆细胞死亡的总体轮廓, 必须对所有这些死亡模式进行多重化验, 这是劳动和成本密集型的。
在这篇文章中, 我们描述了一个快速和 cost-effective one-step 检测的同时评估细胞凋亡, 有丝分裂突变, 和细胞衰老诱导的 IR3,4。在该方法中, 在盖板上生长的细胞用 X 射线照射, 染色 4 ", 6-diamidino-2-吲二盐酸盐 (DAPI)。利用荧光显微镜、细胞凋亡、有丝分裂突变和细胞衰老, 分别根据 DAPI 染色核的特征形态进行鉴定。这种方法将有助于包括筛查克隆细胞死亡剖面在不同的细胞线, 治疗设置和时间点, 目的是调查细胞死亡的机制在靶细胞和条件兴趣.

<table><tbody><tr><td>cover slip</td><td>Matsunami</td><td>C013001</td><td></td></tr><tr><td>paraformaldehyde</td><td>Sigma-Aldrich</td><td>P6148-1KG</td><td></td></tr><tr><td>sucrose</td><td>Sigma-Aldrich</td><td>84097-250G</td><td></td></tr><tr><td>phosphate-buffered saline</td><td>Cosmo Bio</td><td>16232000</td><td></td></tr><tr><td>scalpel</td><td>Kai Medical</td><td>No.20, 520-A</td><td></td></tr><tr><td>35 mm cell culture dish</td><td>Falcon</td><td>353001</td><td></td></tr><tr><td>trypsin</td><td>Wako</td><td>201-16945</td><td></td></tr><tr><td>inverted phase microscope</td><td>Shimazu</td><td>114-909</td><td></td></tr><tr><td>X-ray irradiator</td><td>Faxitron Bioptics</td><td>Faxitron RX-650</td><td></td></tr><tr><td>square culture dish</td><td>Corning</td><td>431110</td><td></td></tr><tr><td>slide glass</td><td>Matsunami</td><td>Pro-11</td><td></td></tr><tr><td>DAPI staining reagent</td><td>Cell Signaling</td><td>8961S</td><td></td></tr><tr><td>fluorescence microscope</td><td>Nikon</td><td>ECLIPSE Ni</td><td></td></tr><tr><td>fluorescence microscope DAPI filter</td><td>Nikon</td><td>U-FUW, BP340-390, BA420IF, DM410</td><td></td></tr><tr><td>fluorescence microscope lens (20&amp;times;)</td><td>Nikon</td><td>Plan Apo &amp;lambda; 20X/0.75</td><td></td></tr><tr><td>fluorescence microscope lens (60&amp;times;, oil)</td><td>Nikon</td><td>Plan Apo &amp;lambda; 60X/1.40 oil</td><td></td></tr><tr><td>oil</td><td>Olympus</td><td>N5218600</td><td></td></tr><tr><td>CCD camera</td><td>Nikon</td><td>DS-Qi2</td><td></td></tr><tr><td>digital image acquisition software</td><td>Nikon</td><td>NIS-Elements Document</td><td></td></tr><tr><td>lens cleaner</td><td>Olympus</td><td>OT0552</td><td></td></tr><tr><td>chloroform</td><td>Wako</td><td>035-02616</td><td></td></tr></tbody></table>

one-step protocol for evaluation of the mode of radiation-induced clonogenic cell death by fluorescence microscopy

电离辐射 (ir) 诱导的克隆细胞死亡的研究对于了解 ir 对恶性肿瘤和正常组织的影响是非常重要的。在这里, 我们描述了一个快速和 cost-effective one-step 测定, 同时评估的主要模式, 克隆细胞死亡的 IR,即, 细胞凋亡, 有丝分裂突变, 细胞衰老。在该方法中, 在盖板上生长的细胞用 X 射线照射, 染色 4 ", 6-diamidino-2-吲二盐酸盐 (DAPI)。采用荧光显微镜、细胞凋亡、有丝分裂突变和细胞衰老的方法, 根据 DAPI 染色核的特征形态进行鉴定。凋亡是由存在的凋亡体 (即, 凝聚和分裂的细胞核)。有丝分裂突变是由表现出两个或多个不同的裂片和微的细胞核的存在所决定的。细胞衰老是由衰老相关的异灶 (即, 细胞核 DNA 包含30-50 明亮, 致密的病灶) 的存在。这种方法允许实验者使用不同的细胞线、治疗设置和/或时间点, 很容易地筛克隆细胞死亡模式, 目的是阐明细胞死亡的机制在目标细胞和感兴趣的条件。

以6个 X 射线为例, 对 U2OS 人骨肉瘤细胞进行了72小时的培养, 并根据该协议进行了 DAPI 染色试验。 图 1显示了与凋亡、有丝分裂突变和使用60×油透镜获得的细胞衰老有关的典型核形态的放大图像。有关克隆细胞死亡的每种模式的特征形态, 请参阅步骤 7.1. 1-7. 1.3。 图 2显示了使用20X 透镜获得的细胞核的全景图像. 放射与 6 X 射线诱发有丝分裂突变频繁, 凋亡频率较低, 细胞衰老很少。以这种方式, 在一个 low-magnification 的领域的检查, 使一个人能够在一个给定的实验环境中一目了然地了解克隆细胞死亡剖面的全貌。 图 3显示量化数据的图形表示形式。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56338/56338fig1.jpg"> 
图 1: DAPI 染色核的放大图像, 显示了细胞凋亡、有丝分裂突变和细胞衰老的典型形态.U2OS 细胞以 6 X 射线照射。72小时后, 细胞被固定和染色的 DAPI。使用60X 油透镜获取核图像。刻度条 = 20 μ m。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56338/56338fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56338/56338fig2.jpg"> 
图 2: X 射线处理的细胞 DAPI 染色细胞核的总览图.U2OS 细胞照射 6 X 射线或模拟辐照。72小时后, 细胞被固定和染色的 DAPI。利用20X 透镜获取细胞核的图像。圆, 凋亡;箭, 有丝分裂的灾难。刻度条 = 50 μ m。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56338/56338fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56338/56338fig3.jpg"> 
图 3: X 射线处理的细胞凋亡、有丝分裂突变和细胞衰老的量化数据.U2OS 细胞照射 6 X 射线或模拟辐照。72小时后, 细胞被固定和染色的 DAPI。利用20X 透镜获取细胞核的图像。从随机场, 总共300核被评价为细胞凋亡 (Ap), 有丝分裂突变 (MC) 和细胞衰老 (Sns)。评价是以三份进行的。平均值显示, 误差线表示标准偏差。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56338/56338fig3large.jpg" style="font-size: 14px;" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about One-step Protocol for Evaluation of the Mode of Radiation-induced Clonogenic Cell Death by Fluorescence Microscopy at JoVE.com

One-step Protocol for Evaluation of the Mode of Radiation-induced Clonogenic Cell Death by Fluorescence Microscopy

荧光显微镜评价辐射诱发克隆细胞死亡模式的一步协议

电离辐射 (ir) 诱导的克隆细胞死亡的研究对于了解 ir 对恶性肿瘤和正常组织的影响是非常重要的。在这里, 我们描述了基于 4 ', 6-diamidino-2-吲二盐酸盐 (DAPI)-荧光显微镜可视化染色核的形态学的红外诱导克隆细胞死亡主要模式的 one-step 测定。

Research on ionizing radiation (IR)-induced clonogenic cell death is important for understanding the effect of IR on malignant tumors and normal tissues. ...

one-step-protocol-for-evaluation-mode-radiation-induced-clonogenic

Nanyang Technological University

Research

JoVE Journal

Cancer Research

11.2K 次观看.  Gunma University Graduate School of Medicine. 电离辐射 (ir) 诱导的克隆细胞死亡的研究对于了解 ir 对恶性肿瘤和正常组织的影响是非常重要的。在这里, 我们描述了基于 4 ', 6-diamidino-2-吲二盐酸盐 (DAPI)-荧光显微镜可视化染色核的形态学的红外诱导克隆细胞死亡主要模式的 one-step 测定。

视频: One-step Protocol for Evaluation of the Mode of Radiation-induced Clonogenic Cell Death by Fluorescence Microscopy

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 ...

科研

癌症研究

Cell Cycle-specific Measurement of &#947;H2AX and Apoptosis After Genotoxic Stress by Flow Cytometry

The presented method or slightly modified versions have been devised to study specific treatment responses and side effects of various anti-cancer treatments as used in clinical oncology. It enables a quantitative and longitudinal analysis of the DNA damage response after genotoxic stress, as induced by radiotherapy and a multitude of anti-cancer drugs. The method covers all stages of the DNA damage response, providing endpoints for induction and repair of DNA double-strand breaks (DSBs), cell cycle arrest and cell death by apoptosis in case of repair failure. Combining these measurements provides information about cell cycle-dependent treatment effects and thus allows an in-depth study of the interplay between cellular proliferation and coping mechanisms against DNA damage. As the effect of many cancer therapeutics including chemotherapeutic agents and ionizing radiation is limited to or strongly varies according to specific cell cycle phases, correlative analyses rely on a robust and feasible method to assess the treatment effects on the DNA in a cell cycle-specific manner. This is not possible with single-endpoint assays and an important advantage of the presented method. The method is not restricted to any particular cell line and has been thoroughly tested in a multitude of tumor and normal tissue cell lines. It can be widely applied as a comprehensive genotoxicity assay in many fields of oncology besides radio-oncology, including environmental risk factor assessment, drug screening and evaluation of genetic instability in tumor cells.

The presented method combines the quantitative analysis of DNA double-strand breaks (DSBs), cell cycle distribution and apoptosis to enable cell cycle-specific evaluation of DSB induction and repair as well as the consequences of repair failure.

细胞周期特异性测量βH2AX和细胞凋亡后热源毒性应力通过流细胞测定

The presented method or slightly modified versions have been devised to study specific treatment responses and side effects of various anti-cancer ...

评估表达 CAR 的 Jurkat 细胞对肿瘤细胞的定量杀伤

Rapid In Vitro Cytotoxicity Evaluation of Jurkat Expressing Chimeric Antigen Receptor using Fluorescent Imaging

Chimeric antigen receptor (CAR) T cells are at the forefront of oncology. A CAR is constructed of a targeting domain (usually a single chain variable fragment, scFv), with an accompanying intra-chain linker, followed by a hinge, transmembrane, and costimulatory domain. Modification of the intra-chain linker and hinge domain can have a significant effect on CAR-mediated killing. Considering the many different options for each part of a CAR construct, there are large numbers of permutations. Making CAR-T cells is a time-consuming and expensive process, and making and testing many constructs is a heavy time and material investment. This protocol describes a platform to rapidly evaluate hinge-optimized CAR constructs in Jurkat cells (CAR-J). Jurkat cells are an immortalized T cell line with high lentivirus uptake, allowing for efficient CAR transduction. Here, we present a platform to rapidly evaluate CAR-J using a fluorescent imager, followed by confirmation of cytolysis in PBMC-derived T cells.

作者聚焦：CAR T 细胞构建体的高通量筛选以增强细胞毒性和免疫记忆 

A protocol to evaluate quantitative tumor cell killing by Jurkat cells expressing chimeric antigen receptor (CAR) targeting single tumor antigen. This protocol can be used as a screening platform for rapid optimization of CAR hinge constructs prior to confirmation in peripheral blood-derived T cells.

使用荧光成像快速体外细胞毒性评估表达嵌合抗原受体的 Jurkat

Chimeric antigen receptor (CAR) T cells are at the forefront of oncology. A CAR is constructed of a targeting domain (usually a single chain variable ...

<meta charset="utf8"></head><body>使用克隆形成测定法检测患者来源的肿瘤类器官的放射反应

Live Imaging to Quantify Cellular Radiosensitivity in Patient-Derived Tumor Organoids

Radiation therapy (RT) is one of the mainstays of modern clinical cancer management. However, not all cancer types are equally sensitive to irradiation, often (but not always) because of differences in the ability of malignant cells to repair oxidative DNA damage as elicited by ionizing rays. Clonogenic assays have been employed for decades to assess the sensitivity of cultured cancer cells to ionizing irradiation, largely because irradiated cancer cells often die in a delayed manner that is difficult to quantify with short-term flow cytometry- or microscopy-assisted techniques. Unfortunately, clonogenic assays cannot be employed as such for more complex tumor models, such as patient-derived tumor organoids (PDTOs). Indeed, irradiating established PDTOs may not necessarily abrogate their growth as multicellular units, unless their stem-like compartment is completely eradicated. Moreover, irradiating PDTO-derived single-cell suspensions may not properly recapitulate the sensitivity of malignant cells to RT in the context of established PDTOs. Here, we detail an adaptation of conventional clonogenic assays that involves exposure of established PDTOs to ionizing radiation, followed by single-cell dissociation, replating in suitable culture conditions and live imaging. Non-irradiated (control) PDTO-derived stem-like cells reform growing PDTOs with a PDTO-specific efficiency, which is negatively influenced by irradiation in a dose-dependent manner. In these conditions, PDTO-forming efficiency and growth rate can be quantified as a measure of radiosensitivity on time-lapse images collected until control PDTOs achieve a predefined space occupancy.

<meta charset="utf8"></head><body>作者聚焦:用于推进癌症研究和个性化医疗的放射治疗和克隆形成检测

Here, we detail a straightforward live imaging approach for quantifying the sensitivity of patient-derived tumor organoids to ionizing radiation.

实时成像量化患者来源的肿瘤类器官中的细胞放射敏感性

Radiation therapy (RT) is one of the mainstays of modern clinical cancer management. However, not all cancer types are equally sensitive to irradiation, ...

Clonogenic Assay: Adherent Cells

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. Apart from documenting the method, the initial landmark study generated the first radiation-dose response curve for X-ray irradiated mammalian (HeLa) cells in culture1. Basically, the clonogenic assay enables an assessment of the differences in reproductive viability (capacity of cells to produce progeny; i.e. a single cell to form a colony of 50 or more cells) between control untreated cells and cells that have undergone various treatments such as exposure to ionising radiation, various chemical compounds (e.g. cytotoxic agents) or in other cases genetic manipulation. The assay has become the most widely accepted technique in radiation biology and has been widely used for evaluating the radiation sensitivity of different cell lines. Further, the clonogenic assay is commonly used for monitoring the efficacy of radiation modifying compounds and for determining the effects of cytotoxic agents and other anti-cancer therapeutics on colony forming ability, in different cell lines. A typical clonogenic survival experiment using adherent cells lines involves three distinct components, 1) treatment of the cell monolayer in tissue culture flasks, 2) preparation of single cell suspensions and plating an appropriate number of cells in petri dishes and 3) fixing and staining colonies following a relevant incubation period, which could range from 1-3 weeks, depending on the cell line. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cell lines with the use of an immortalized human keratinocyte cell line (FEP-1811)2. Also, our aims are to describe common features of clonogenic assays including calculation of the plating efficiency and survival fractions after exposure of cells to radiation, and to exemplify modification of radiation-response with the use of a natural antioxidant formulation.

The applicability of the clonogenic assay for evaluating reproductive viability has been established for more than 50 years. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cells.

克隆形成含量:贴壁细胞

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. ...

生物学

Preparation of Cell-lines for Conditional Knockdown of Gene Expression and Measurement of the Knockdown Effects on E4orf4-Induced Cell Death

Functional inactivation of gene expression in mammalian cells is crucial for the study of the contribution of a protein of interest to various pathways1,2. However, conditional knockdown of gene expression is required in cases when constitutive knockdown is not tolerated by cells for a long period of time3-5. Here we describe a protocol for preparation of cell lines allowing conditional knockdown of subunits of the ACF chromatin remodeling factor. These cell lines facilitate the determination of the contribution of ACF to induction of cell death by the adenovirus E4orf4 protein6. Sequences encoding short hairpin RNAs for the Acf1 and SNF2h subunits of the ACF chromatin remodeling factor were cloned next to a doxycycline-inducible promoter in a plasmid also containing a gene for the neomycin resistance gene. Neomycin-resistant cell clones were selected in the presence of G418 and isolated. The resulting cell lines were induced by doxycycline treatment, and once Acf1 or SNF2h expression levels were reduced, the cells were transfected with a plasmid encoding E4orf4 or an empty vector. To confirm the specific effect of the shRNA constructs, Acf1 or SNF2h protein levels were restored to WT levels by cotransfection with a plasmid expressing Acf1 or SNF2h which were rendered resistant to the shRNA by introduction of silent mutations. The ability of E4orf4 to induce cell death in the various samples was determined by a DAPI assay, in which the frequency of appearance of nuclei with apoptotic morphologies in the transfected cell population was measured7-9.
The protocol described here can be utilized for determination of the functional contribution of various proteins to induction of cell death by their protein partners in cases when constitutive knockdown may be cell lethal.

Contribution of the ACF chromatin remodeling factor to E4orf4-induced cell death was measured. The protocol includes selection of cell clones in which doxycycline treatment induces conditional knockdown of the ACF subunits Acf1 and SNF2h, and use of the DAPI assay to measure E4orf4-induced cell death in the inducible cell lines.

荧光显微镜评价辐射诱发克隆细胞死亡模式的一步协议

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