这项工作是支持的，部分由生物学卓越研究[授权号1P20GM109005]，阿肯色空间格兰特财团通过美国国家航空和航天局[授权号NNX15AK32A]卫生中心的国家研究所和美国国家职业安全和健康研究所（NIOSH）授权号码2T420H008436。笔者想感谢克里斯托弗Fettes校对和编辑这篇稿子。

1.颗粒收集和准备<ol><li> 高容量总悬浮微粒采样器的校准 <ol><li>断开质量流量控制器的采样器马达和马达连接到一个稳定的交流电源（ 图1）。 注:在此过程中不使用的过滤器。 </li><li>校准器安装孔和顶部装载转接板到采样。拧紧顶部装载适配器压紧螺母牢固，以确保没有空气泄漏是否存在。允许采样马达升温到其正常的操作温度（约10-15分钟）。 </li><li>覆盖与手口在管口和测压管顶部的孔进行泄漏测试。侦听由逃逸的空中发出高亢的尖叫声。由于存在泄漏如果听到声音，再拧紧顶部装载适配器压紧螺母。如果声音是较低时，泄漏是靠近在sys其他垫圈之一透射电镜。 </li><li>水压力计的一侧连接到压力抽头上用橡胶真空管孔口的一侧。离开压力计通向大气的相对侧。保持一个压力计垂直，以确保准确的读数。攻连续流动记录的背面，将有助于居中笔，并提供准确的读数。 </li><li>重复步骤1.1.2使用从30至60英尺/分钟（大约每5-6英尺3 /分钟），代表工作流率5流速。调整对可变孔旋钮五个不同位置，采取五种不同的读数。 </li><li>记录周围空气的温度，环境大气压，取样序号，孔口序列号，孔口斜率和截距与日期最后认证，日期，场地的位置，和操作者的对校准片缩写。 </li></ol></li><li> 总悬浮颗粒物样品采集和重分析 <ol><li>一个包裹8"×10"，在铝箔的两层石英纤维过滤器。放置在炉中的卷绕的过滤器，并设置在550℃的温度。烤滤波器为至少4小时。让炉冷并检索滤波器。放置过滤器与碳酸钙的干燥器。 </li><li>权衡为10微克精度微量天平过滤器。在一天重复测量三次。单独的测量应在25微克。如果不这样实现的，被包装的过滤器返回到干燥器并重复此过程次日。 </li><li>打开盖子住所（ 图2）。松开四个蝶形螺母，使铜螺栓和垫圈，翻下出的方式删除过滤器保持框。从打开铝箔过滤器，并使用清洁镊子小心地居中，粗糙的一面朝上，支持屏幕上。正确对齐屏幕上的过滤器。 </li><li>在画面上放置框架和保护它黄铜螺栓和垫圈而施加足够的压力，以避免在边缘处的空气泄漏。擦掉过滤器支架周围的任何灰尘堆积用干净的布。关闭住所盖子仔细并固定。 </li><li>确保所有线均已插入其合适的插座插座及鼓风机马达压力水龙头，连续流动记录之间的管道连接。 </li><li>准备流量记录。按下笔臂升降，以提高笔尖，并插入一个新的图表。对齐图表记录仪的驱动毂的选项卡，然后按拇指，以降低图表上中心枢纽。通过直至图表正确的时间与时间索引指针对准旋转驱动轮毂顺时针设定的时间。 </li><li>手动切换的采样，并开始收集。监控记录，以确保它是正确着墨和经过时间指示器，以确保其工作正常。 </li><li>在采样周期结束时，关掉采样器，记录的经过时间，并检索该图表。删除框架，露出过滤器。轻轻保持它在与镊子的末端移除所述支撑屏幕的露出滤波器。纵向折叠过滤器，使样品接触的样品两次，在原有的铝箔包裹。 </li><li>与碳酸钙称重之前存储过滤干燥器中至少24小时。重复步骤1.2.2取样后测量过滤器的重量。放置过滤器在一拉链塑料袋中并储存在-80℃直至分析。 </li></ol></li><li> 收集的总悬浮颗粒物采样分析 <ol><li>检索存储的过滤器，展开铝箔和一个干净的表面特氟龙转移过滤器。使用镊子和手术刀，切开过滤器外（白色）的一部分，处理。 <ol><li>接着，切断该过滤器在0.5"×0.5"件和地点片在100毫升烧瓶中。加入50mL超纯水。放置烧瓶在超声浴中并在33±3千赫超声60分钟。 </li></ol></li><li>通过0.45μm聚丙烯注射器过滤器的100毫升圆底烧瓶中过滤提取物。在50℃下蒸发使用压力下的旋转蒸发仪中的水，直至约5ml提取物保留在烧瓶中。 <ol><li>蒸发剩余的水在已知重量的15毫升梨形瓶中。权衡与干提取物的烧瓶中，并记录干提取物的质量。 </li></ol></li><li>通过超声处理15分钟溶解用400μlD 2 O中的干燥样品，并转移到一个5毫米的标准NMR管。在12,000 xg离心离心5分钟，在室温下，以除去任何未溶解的物质。 </li><li>重复用300μlD 2的O.添加10微升NaN 3的1.4％（终浓度:0.02％重量/体积）在NMR管。添加10微升（32微克）总悬浮颗粒物（TSP）〜D4（3.2毫克/毫升）的在NMR管D 2 O中（终浓度0.258毫摩尔）。 </li><li>通过收购在600.17兆赫运行的核磁共振波谱仪1核磁共振光谱。 <ol><li>将NMR管进入三重共振冷冻探针。校准仪器按以下顺序:锁，调整垫片，90°脉冲的长度校准器和接收增益调整。 </li><li>测量在25℃的光谱，使用在数字四路检测模式180水选择性脉冲，8,192扫描，4虚设扫描，8012.57赫兹的扫描宽度的1个D激励雕刻顺序，频率为水信号抑制设定在4.70 ppm的，1.70偏移秒的采集时间，32,768时域数据点（TD），1毫秒的时间梯度（P16），后梯度（D16）100微秒恢复延迟。 </li></ol></li><li>过程1通过2（65,536个数据点）的一个因素施加1赫兹和零填充的谱线增宽H-NMR光谱。手动校正阶段和基线和整合。在0.0 ppm的设置TSP-D4高峰。 </li></ol></li></ol> 2.粒子曝光<ol><li> 文化成立 <ol><li>含有10％胎牛血清（FBS）和青霉素 - 链霉素溶液（100单位/毫升），0.25％胰蛋白酶溶液和钙镁自由磷酸盐缓冲盐水（PBS）在水浴（37℃）预热完全生长培养基为培养开始前至少30分钟。 </li><li>取出从含有培养的RAW264.7细胞，5％CO 2培养箱一个T75烧瓶中并用2ml的预温热的PBS两次洗涤细胞。 </li><li>逐出的组织培养容器中的用1ml预温热胰蛋白酶溶液，接着刮底部粘附RAW264.7细胞。 </li><li>收集来自组织培养容器中的细胞，并通过反复吹打单细胞悬浮液。算用锥虫蓝和板细胞的数量在预先温热完整的媒体100毫米组织培养皿一个8000 /厘米2（500,000个细胞总数）密度。调节介质体积10毫升50,000个细胞的终浓度每毫升。 注:加倍板的数量，如果也进行DNA甲基化分析。 </li><li>放置新镀RAW264.7细胞回在5％CO 2培养箱中在37℃，并允许以24小时连接。 </li><li>此外，在生物安全柜，过滤悬浮颗粒。例如，以0.5毫克/毫升无菌PBS为5微克/毫升的治疗剂量的浓度稀释的颗粒，并以5毫克/毫升为50微克/毫升的治疗剂量。稀释可以事先准备最多一个月并储存在-80℃。 </li></ol></li><li> 治疗用微粒物 <ol><li>取出从5％CO 2培养箱的细胞和使用用于细胞生长，细胞形态，培养条件，和污染在倒置显微镜下10倍放大镜检查。之一应该期望具有至少30％汇合的细胞在这个阶段。 </li><li>无菌，使用消毒吸管吸媒体并用2ml 37℃的PBS洗涤培养容器两次以完全去除媒体和浮动或死亡的细胞。 </li><li>添加的颗粒物质预先确定的剂量，用新鲜培养基在培养容器和在5％CO 2培养箱中 ，在37°C孵育所需的时间。在这项研究中，治疗RAW264.7细胞用PM 72小时为并行用于并发基因毒性和epigenotoxic分析曝光。 注:细胞毒性可在细胞遗传学分析的质量产生负面影响。因此，如细胞毒性，DNA甲基化，Western印迹，或基因表达分析的附加测定在处理过的细胞中通常进行的。暴露的细胞所描述和与期望的特定测定法进行。没有经验的用户也可能希望包括阳性对照，如甲基甲磺酸，以测试他们检测的CA的能力。 </li></ol></li></ol> 3.细胞遗传学分析<ol><li> 治疗逮捕在中期细胞 <ol><li>暖秋水仙碱这样lution（10微克/毫升）在37℃水浴中30分钟。擦拭用70％酒精盖生物安全柜下，以尽量减少污染的风险</li><li>完全无菌删除媒体并用2ml预热的PBS两次洗培养容器。 </li><li>添加预热的新鲜培养基（2毫升培养基/ 10 6个细胞）中含有培养容器秋水仙胺溶液（5微升/ ml）和孵育2小时。 </li></ol></li><li> 收获细胞 <ol><li>温暖的胰蛋白酶溶液和PBS的水浴中保持在37℃30分钟。从该步骤起，就没有必要保持无菌状态。 </li><li>彻底去除媒体，用2毫升37℃PBS洗涤培养容器两次，加预热的胰蛋白酶溶液所需体积（通常为2毫升至60mm培养盘或T25瓶和4毫升100毫米的菜或T75烧瓶），以及培养容器放回在5％CO 2培养箱中在37℃下1分钟。 </li><li>德Ë出培养容器，分离用刮刀的细胞，并加入10毫升的PBS以停止胰蛋白酶作用。 </li><li>移液器上下至少十倍，使单细胞悬浮液并转移细胞在15ml圆锥形底离心管中。 </li><li>在室温下摆动出来离心机在400×g离心5分钟离心细胞悬浮液。 </li><li>除去上清液，通过轻轻敲击打破细胞沉淀，加入10毫升的PBS，并再次离心五分钟。 </li></ol></li><li> 低渗处理和固定 <ol><li>在37℃水中至少30分钟浴预暖0.075 1M氢氯化物溶液。 </li><li>而不干扰细胞沉淀除去上清液，留下大约为0.5毫升的PBS。 </li><li>轻轻打破细胞沉淀，并用P200移液管使单细胞悬浮液。 </li><li>通过轻轻摇动滴加入4毫升预热氯化钾溶液滴。 </li><li>孵育低渗P中的细胞在37℃水浴中20分钟otassium氯化物溶液。 </li><li>加入3份的甲醇与1部分冰醋酸做出新的acetomethanol解决方案。保持这个新鲜配制固定液在室温下。 </li><li>低渗处理后，添加固定剂的等体积（4毫升）中的管和通过反转所述管将溶液轻轻混匀。 </li><li>离心在400 xg离心在室温下5分钟，并除去上清液。在此阶段细胞沉淀尺寸增大，变得发白和更明显。 </li><li>去除上清液，加入4毫升的新鲜固定液，并保持在室温下30分钟。 </li><li>离心机在400 XG 5分钟，除去上清液，并给出两个洗涤用4ml固定液。 </li></ol></li><li> 滑动清洗和中期扩展准备 <ol><li>完全在10％的液体洗涤剂浸渍的玻璃载玻片30分钟。 </li><li>彻底冲洗载玻片在冷的流动的自来水30分钟。 </li><li>凛本身用蒸馏水滑动三次以彻底除去洗涤剂，在蒸馏水浸泡，并在冰箱中以备将来使用存储。 注:清洁幻灯片便于使用前均匀传播，提高质量的中期。 </li><li>滴上冷却湿滑板10微升细胞悬浮液，并允许它空气中在室温下干燥过夜。 </li></ol></li><li> 染色和安装 <ol><li>用PBS的一个部分混合姬姆萨染色解决方案的一部分，准备50毫升姬姆萨染色解决方案，并倒在一个罐子科普林。 </li><li>浸入染色溶液中的玻片20分钟。 </li><li>冲洗载玻片在蒸馏水以除去过量染色剂和立即干燥用吹风机的幻灯片。 </li><li>留在室温下将载玻片过夜。 </li><li>适用于滑动安装平台的单个下降，轻轻地放在一个盖玻片安装介质上，允许介质慢慢滑动传播，并取出用纸巾过量介质。允许安装型滑轨，以至少1小时移动或在显微镜下观察，以避免盖玻片运动之前干燥。 注意:要仔细放置盖玻片而不在该步骤中形成的任何气泡是非常重要的。建议不要使用过多的热量，以消除气泡。 </li></ol></li><li> 显微观察和畸变类型 <ol><li>检查结构的CA在中期使用高质量的亮场显微镜放大100倍的利差。 </li><li>拿下至少50至100中期利差为诱导大量的CA治疗各治疗组。对于较小的影响大小，高达300中期利差的分析建议。 </li></ol></li></ol>

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Sci. 148 (2), 473-487 (2015).</li><li>Ehrlich, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+instability+in+cancer+development.">Genomic instability in cancer development.</a> Genome Instability in Cancer Development. , 363-392 (2005).</li><li>Michael, S., Montag, M., Dott, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pro-inflammatory+effects+and+oxidative+stress+in+lung+macrophages+and+epithelial+cells+induced+by+ambient+particulate+matter.">Pro-inflammatory effects and oxidative stress in lung macrophages and epithelial cells induced by ambient particulate matter.</a> Environ Pollut. 183, 19-29 (2013).</li><li>Li, N., 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=Induction+of+heme+oxygenase-1+expression+in+macrophages+by+diesel+exhaust+particle+chemicals+and+quinones+via+the+antioxidant-responsive+element.">Induction of heme oxygenase-1 expression in macrophages by diesel exhaust particle chemicals and quinones via the antioxidant-responsive element.</a> J Immunol. 165 (6), 3393-3401 (2000).</li><li>Pathak, R., Ramakumar, A., Subramanian, U., Prasanna, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+radio-sensitivities+of+human+chromosomes+1+and+2+in+one+donor+in+interphase-+and+metaphase-spreads+after+60Co+gamma-irradiation.">Differential radio-sensitivities of human chromosomes 1 and 2 in one donor in interphase- and metaphase-spreads after 60Co gamma-irradiation.</a> BMC Med. Phys. 9, 6649 (2009).</li><li>Pathak, R., Dey, S. K., Sarma, A., Khuda-Bukhsh, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genotoxic+effects+in+M5+cells+and+Chinese+hamster+V79+cells+after+exposure+to+7+Li-beam+(LET=+60keV/&#181;m)+and+correlation+of+their+survival+dynamics+to+nuclear+damages+and+cell+death.">Genotoxic effects in M5 cells and Chinese hamster V79 cells after exposure to 7 Li-beam (LET= 60keV/&#181;m) and correlation of their survival dynamics to nuclear damages and cell death.</a> Mutat. Res. Genet. Toxicol. Environ. Mutagen. 628 (1), 56-66 (2007).</li><li>Pathak, R., Dey, S. K., Sarma, A., Khuda-Bukhsh, A. 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The authors have nothing to disclose.

细胞遗传学研究或数字或染色体，主要是在中期扩展的结构，微观分析，为预测，风险评估和治疗各种疾病的关键信息。它现在公认该细胞遗传学异常与几种疾病，包括癌症的进展和发展相关。迄今为止，中国科学院已经在所有主要类型的肿瘤中发现。可诱导各种类型的细胞遗传学改变16的主要外部危险因素之一-的CA可以自发或通过外部或内部的刺激，如电离辐射（IR）的出现。 CA的辅助分析所吸收的辐射剂量的评估（所谓的细胞遗传学辐射生物剂量）。此外，频率和特定像差类型的分析还提供了用于确定辐射的品质的重要信息被吸收17,18。细胞遗传学研究提供了我的直接图像对DNA损伤剂的MPACT。这是在对比的间接技术，如彗星试验。然而，细胞遗传学研究已经因为众多动手参与步骤部分辐射之外的领域得到充分利用。 虽然接触有机存在于PM碳众所周知，我们的健康产生不利影响，一些研究报告的生物效应包括其组成9。这是由于需要劳动密集型的提取，加工，浓度和衍生协议的昂贵和麻烦的色谱方法，而它们只提供对于每个时间非常特定类型的化合物的一个小的子集的见解。此外，该协议导致在基本操作或原样品的破坏，因此，不能进行进一步的测试。核磁共振（NMR）分析的优点包括核磁共振是一种非破坏性的方法;它需要STE的数量非常有限PS（提取，浓缩）分析前，和高频核磁共振仪器（400 MHz至900兆赫）在许多可用的，如果不是全部，核心设施内的大学。大气气溶胶核磁共振研究的全面审查，最近还发表了19。最后，由于其提供的结构信息和组之间的键的能力，核磁共振优于其他光谱测定的方法，如红外光谱，紫外 - 可见和拉曼光谱，只有提供官能团的类型的数据。如果需要的话，PM的形态特征可以另外通过使用扫描电子显微镜执行以NMR谱。 即使从单个位置，PM的组合物收集可以根据一年中的时间而变化。足够大的量的材料的应处理，并保持在-80℃下进行重复实验或互补测定。步骤2.1.1通过3.1.3应在BIOS无菌条件下进行afety内阁。如果观察到污染或破坏细胞形态，不符合协议的其余部分继续进行。胰蛋白酶处理时间需要根据细胞类型进行调整。这里，巨噬细胞是很粘附的，我们已经发现，1分钟胰蛋白酶和刮的组合效果最好，但单独胰蛋白酶进行5分钟作品大多数细胞类型。低渗处理时间也取决于细胞类型而不同，并且需要被标准化。秋水仙胺的剂量和处理时间是用于染色体制备的最重要的参数;过剂量可杀死细胞或失速细胞周期。我们在这里描述的条件RAW264.7细胞，以及所需的其它细胞类型优化。未能找到在这个步骤理想的条件下可以在有丝分裂指数产生不利的影响，以及染色体的形态，影响的结果。此外，还建议评估PM的毒性之前细胞遗传学分析水平，并选择相应的剂量。 在体外毒理学评估由所使用的细胞，暴露量值，和颗粒浓度的类型的限制。此外，在该研究中，我们使用的PM的水溶性提取物，因此，不溶于水的物质，以引起细胞遗传学异常的潜在可能不被占。其他限制与的"多态变体"的存在相关 - 在着丝粒区和染色体的短臂的变化;一些亚微观或可能被误解隐蔽重排和复杂核型的存在。 这里，我们表明，引入细胞遗传学分析，可以在与DNA PM引起的损伤的识别有助于并且可以潜在地作为在引起的暴露于环境的PM健康影响评估的预测端点。我们的研究是，就我们所知，第一个表示的CA响应于PM。这些结果的复制是希望证实我们的发现。该协议也可以适于评估几乎任何类型的疑似DNA损伤剂。

据估计，暴露于颗粒物（PM）导致每年超过300万过量死亡，主要是从心肺疾病和肺癌1。的确，PM被确认为致癌由国际癌症研究机构（组1）人类，如肺癌的暴露的水平提高到下午的风险增加已经显示2。有趣的是，几乎所有的肿瘤细胞海港数值和/或结构染色体异常。颗粒有机碳是一种变型的，复杂的，和非均相混合物，其组成和大小分布取决于排放以及物理和化学变化。它占来自城市的PM2.5质量的35-55％（PM 2.5微米尺寸和更小）以及农村和大陆背景的PM 2.5质量3,4的60％以上。水溶性部分占有机气溶胶的30-90％。大量有机化合物已经确定，包括脂族烃，多环芳烃，和它们的氧化和硝化的衍生物，脂族醛和醇，游离脂肪酸和它们的盐，二羧酸，多官能的化合物，蛋白质和类腐殖质大分子（HULIS）使用色谱法加上质谱技术5-8。这些化合物代表微粒的有机碳小于10-20％，因此大多数的有机碳是未知9。 实验证据表明，细胞毒性，氧化应激和炎症中涉及的PM-有关的病理状态的发展。最近已显示，但是，暴露于PM也导致一些外遗传改变的，包括在重复元件的DNA甲基化的改变，在这两个实验在体外系统以及在人受试者10-12。特别感兴趣的是的效果PM卫星DNA - 主要和次要的卫星 - 这是在围绕染色体着丝粒的异色地区发现的。结果表明，这些效果可能是由自然持久的，因为他们可以在至少72小时曝光12后进行检测。改变DNA甲基化，特别是围绕着丝粒，可以在细胞分裂过程导致的卫星DNA的mRNA转录物，折衷染色体完整性累积，并随后导致的各种病理状态13的发展。 为CA的分析所造成的PM后生改建的终点细胞遗传学方法的适应性，因此具有很高的重要性。在这里，我们报告使用小鼠巨噬细胞RAW264.7模型系统中的CA 体外照射和分析环境PM收集和准备的办法。巨噬细胞包括防御的第一线对吸入异物，因此，叔他的细胞系用作建立和粒子毒理学11，12，14，15中的最常用的模型。

<table border="0" cellpadding="0" cellspacing="0" width="726">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Total suspended particulate sampler</td>
			<td style="width:179px;">Tisch Environmental</td>
			<td style="width:119px;">TE-5170</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:261px;">Bruker Avance III NMR spectrometer&#160;</td>
			<td style="width:179px;">Bruker</td>
			<td style="width:119px;">NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">TopSpin 3.5/pl2 software</td>
			<td style="width:179px;">Bruker</td>
			<td style="width:119px;">NA</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:261px;">ACD/NMR Processor Academic Edition</td>
			<td style="width:179px;">ACD/Labs</td>
			<td style="width:119px;">NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RAW264.7 murine macrophages</td>
			<td style="width:179px;">ATCC</td>
			<td style="width:119px;">ATCC TIB-71</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:261px;">High glucose DMEM GlutaMAX media</td>
			<td style="width:179px;">ThermoFisher</td>
			<td align="right" style="width:119px;">10569010</td>
			<td>Warm in a 37&#176;C waterbath before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Fetal Bovine Serum</td>
			<td style="width:179px;">ThermoFisher</td>
			<td align="right" style="width:119px;">16000044</td>
			<td>Warm in a 37&#176;C waterbath before use</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:261px;">Penicillin-Streptomycin (10,000 U/mL)</td>
			<td style="width:179px;">ThermoFisher</td>
			<td align="right" style="width:119px;">15140163</td>
			<td>Warm in a 37&#176;C waterbath before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Trypsin-EDTA (0.25%)</td>
			<td style="width:179px;">ThermoFisher</td>
			<td align="right" style="width:119px;">25200056</td>
			<td>Warm in a 37&#176;C waterbath before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">PBS, pH 7.4</td>
			<td style="width:179px;">ThermoFisher</td>
			<td align="right" style="width:119px;">10010049</td>
			<td>Warm in a 37&#176;C waterbath before use</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:261px;">KaryoMAX Colcemid Solution in PBS</td>
			<td style="width:179px;">ThermoFisher</td>
			<td align="right" style="width:119px;">15212012</td>
			<td>Warm in a 37&#176;C waterbath before use</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:261px;">KaryoMAX Potassium Chloride Solution</td>
			<td style="width:179px;">ThermoFisher</td>
			<td align="right" style="width:119px;">10575090</td>
			<td>Warm in a 37&#176;C waterbath before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Methanol (HPLC)</td>
			<td style="width:179px;">Fisher Scientific</td>
			<td style="width:119px;">A452N1-19</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Acetic Acid, Glacial</td>
			<td style="width:179px;">Fisher Scientific</td>
			<td style="width:119px;">BP1185-500</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:261px;">Decon Contrad 70 Liquid Detergent</td>
			<td style="width:179px;">Fisher Scientific</td>
			<td style="width:119px;">04-355-1</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:261px;">Wright and Wright-Giemsa Stain Solutions</td>
			<td style="width:179px;">Fisher Scientific</td>
			<td style="width:119px;">23-200733</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Permount Mounting Medium</td>
			<td style="width:179px;">Fisher Scientific</td>
			<td style="width:119px;">SP15-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Axio Imager 2</td>
			<td style="width:179px;">Zeiss</td>
			<td style="width:119px;">NA</td>
			<td></td>
		</tr>
	</tbody>
</table>

analysis of the ambient particulate matter-induced chromosomal aberrations using an in vitro system

暴露于颗粒物质（PM）是一个主要的全球健康问题，这可能会损坏的各种细胞组分，包括核遗传物质。为了评估PM的核遗传完整性的影响，染色体结构畸变的得分在小鼠巨噬细胞RAW264.7细胞的中期扩展。 PM被从环境空气中具有高体积总悬浮颗粒采样收集。所收集的材料被溶解，并过滤以保持水溶性，细部。所述颗粒的特征在于用于通过核磁共振（NMR）谱的化学组成。不同浓度的颗粒悬浮液中加入到RAW264.7小鼠巨噬细胞的体外培养72小时的总暴露时间，与未处理的对照细胞一起。在曝光结束后，文化与秋水仙胺治疗逮捕细胞分裂中期。细胞，然后收获，用低渗溶液，定格在acetomethanol，D处理ropped到玻片上，最后用吉姆萨溶液染色。幻灯片进行检查，以评估使用亮场显微镜的结构染色体畸变（CA）的分裂中期利差在放大1000倍。 50至100中期扩展被打进每个治疗组。该技术适于检测结构染色体畸变（CA）的，如染色单体型断裂，染色单体型交流，偏心片段，双着丝粒和环染色体，双分钟，核内复制，和罗伯逊易位体外暴露于PM之后的。这是一个行之有效的细胞遗传学端点后天的改变相关联的有效方法。

极大，应注意在选择TSP采样器的位置，以及当进行收集一年中的时间。的化学组成，以及颗粒的尺寸可以基本上影响结果。收集的材料应该是对白色滤波器可见。普通鼠标中期扩展将有40偏心染色体。该技术的目的是要证明在异常染色体中处理的细胞与对照相比的比例的变化（或没有）。那么这种改变可以被量化（染色体异常的数量）和合格（类型异常）。 正常小鼠中期扩展将有40条染色体偏心（ 图3A）。用颗粒物处理诱导， 如图3所示的各种CA，如染色单体断裂（3B），无中心片段（ <str翁&gt; 3C）中，环染色体（3D），双着丝粒染色体（3E），双分钟（3F）， -罗伯逊易位（3G），和核内复制（3H）。对于全部结果，请参考我们发表的研究报告12。 <img alt="图1" src="/files/ftp_upload/54969/54969fig1.jpg" /> 图1.高容量总悬浮颗粒物（TSP）采样。图为安装在屋顶在城市地区收集环境颗粒采样TSP。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54969/54969fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54969/54969fig2.jpg" /> 采用高容量TSP秒图2.加载石英纤维过滤器收集如下更加宽裕。安装在TSP采样滤波器特写，使用后。注意，过滤器的颜色从白色变成灰色。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54969/54969fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54969/54969fig3.jpg" /> 图3.显微照片示出了用不同浓度的颗粒物质处理后的不同类型在RAW264.7细胞诱导结构畸变的代表性例子。像差由箭头指示。 （ 一 ）正常中期扩展与40近端着丝粒染色体，（B）染色单体型断裂（CTB），（C）偏心片段（acentrics），（D）环状染色体，（E）双着丝粒染色体（DIC），（F）双分（Dimn），（ <stro纳克&gt; G）的罗伯逊易位，和（H）核内复制（复制的染色体保持在一起）。原始放大倍数100X，比例尺= 5微米。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54969/54969fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Analysis of the Ambient Particulate Matter-induced Chromosomal Aberrations Using an In Vitro System at JoVE.com

Analysis of the Ambient Particulate Matter-induced Chromosomal Aberrations Using an In Vitro System

该协议与周围空气的微粒物质处理后描述了在RAW264.7小鼠巨噬细胞体外量化和染色体畸变的表征技术。

使用了大气颗粒物引起的染色体畸变分析<em&gt;体外</em&gt;系统

Exposure to particulate matter (PM) is a major world health concern, which may damage various cellular components, including the nuclear genetic material. ...

analysis-ambient-particulate-matter-induced-chromosomal-aberrations

Nanyang Technological University

Research

JoVE Journal

Genetics

Analysis of the Ambient Particulate Matter-induced Chromosomal Aberrations Using an In Vitro System

8.6K 次观看.  University of Arkansas for Medical Sciences. 该协议与周围空气的微粒物质处理后描述了在RAW264.7小鼠巨噬细胞体外量化和染色体畸变的表征技术。 

视频: Analysis of the Ambient Particulate Matter-induced Chromosomal Aberrations Using an In Vitro System

In Vitro Transcription Assays and Their Application in Drug Discovery

In vitro transcription assays have been developed and widely used for many years to study the molecular mechanisms involved in transcription. This process requires multi-subunit DNA-dependent RNA polymerase (RNAP) and a series of transcription factors that act to modulate the activity of RNAP during gene expression. Sequencing gel electrophoresis of radiolabeled transcripts is used to provide detailed mechanistic information on how transcription proceeds and what parameters can affect it. In this paper we describe the protocol to study how the essential elongation factor NusA regulates transcriptional pausing, as well as a method to identify an antibacterial agent targeting transcription initiation through inhibition of RNAP holoenzyme formation. These methods can be used a as platform for the development of additional approaches to explore the mechanism of action of the transcription factors which still remain unclear, as well as new antibacterial agents targeting transcription which is an underutilized drug target in antibiotic research and development.

In Vitro Transcription Assays and Their Application in Drug Discovery

In this manuscript, we describe a protocol to functionally examine transcription and the inhibitory activity of antibacterial agents targeting bacterial transcription.

体外转录测定及其在药物研究中的应用

In vitro transcription assays have been developed and widely used for many years to study the molecular mechanisms involved in transcription. This process ...

科研

遗传学

Detection of Inter-chromosomal Stable Aberrations by Multiple Fluorescence In Situ Hybridization (mFISH) and Spectral Karyotyping (SKY) in Irradiated Mice

Ionizing radiation (IR) induces numerous stable and unstable chromosomal aberrations. Unstable aberrations, where chromosome morphology is substantially compromised, can easily be identified by conventional chromosome staining techniques. However, detection of stable aberrations, which involve exchange or translocation of genetic materials without considerable modification in the chromosome morphology, requires sophisticated chromosome painting techniques that rely on in situ hybridization of fluorescently labeled DNA probes, a chromosome painting technique popularly known as fluorescence in situ hybridization (FISH). FISH probes can be specific for whole chromosome/s or precise sub-region on chromosome/s. The method not only allows visualization of stable aberrations, but it can also allow detection of the chromosome/s or specific DNA sequence/s involved in a particular aberration formation. A variety of chromosome painting techniques are available in cytogenetics; here two highly sensitive methods, multiple fluorescence in situ hybridization (mFISH) and spectral karyotyping (SKY), are discussed to identify inter-chromosomal stable aberrations that form in the bone marrow cells of mice after exposure to total body irradiation. Although both techniques rely on fluorescent labeled DNA probes, the method of detection and the process of image acquisition of the fluorescent signals are different. These two techniques have been used in various research areas, such as radiation biology, cancer cytogenetics, retrospective radiation biodosimetry, clinical cytogenetics, evolutionary cytogenetics, and comparative cytogenetics.

Detection of Inter-chromosomal Stable Aberrations by Multiple Fluorescence In Situ Hybridization mFISH and Spectral Karyotyping SKY in Irradiated Mice

The present protocol describes the usefulness of multiple fluorescence in situ hybridization (mFISH) and spectral karyotyping (SKY) in identifying inter-chromosomal stable aberrations in the bone marrow cells of mice after exposure to total body irradiation.

通过多色荧光跨染色体畸变稳定的检测<em&gt;原位</em&gt;杂交（渔业部）和光谱核型分析（SKY）的照射小鼠

Ionizing radiation (IR) induces numerous stable and unstable chromosomal aberrations. Unstable aberrations, where chromosome morphology is substantially ...

Analysis of the c-KIT Ligand Promoter Using Chromatin Immunoprecipitation

Multiple cellular processes, including DNA replication and repair, DNA recombination, and gene expression, require interactions between proteins and DNA. Therefore, DNA-protein interactions regulate multiple physiological, pathophysiological, and biological functions, such as cell differentiation, cell proliferation, cell cycle control, chromosome stability, epigenetic gene regulation, and cell transformation. In eukaryotic cells, the DNA interacts with histone and nonhistone proteins and is condensed into chromatin. Several technical tools can be used to analyze DNA-protein interactions, such as the Electrophoresis (gel) Mobility Shift Assay (EMSA) and DNase I footprinting. However, these techniques analyze the protein-DNA interaction in vitro, not within the cellular context. Chromatin immunoprecipitation (ChIP) is a technique that captures proteins at their specific DNA binding sites, thereby allowing for the identification of DNA-protein interactions within their chromatin context. It is done by fixation&#160;of the DNA-protein interaction, followed by&#160;immunoprecipitation of the protein of interest. Subsequently, the genomic site that the protein was bound to is characterized. Here, we describe and discuss ChIP and demonstrate its analytical value for the identification of the Transforming Growth Factor-&#946; (TGF-&#946;)-induced binding of the transcription factor SMAD2 to SMAD Binding Elements (SBE) within the promoter region of the tyrosine-protein kinase Kit (c-KIT) receptor ligand Stem Cell Factor (SCF).

DNA-protein interactions are essential for multiple biological processes. During the evaluation of cellular functions, the analysis of DNA-protein interactions is indispensable for understanding gene regulation. Chromatin immunoprecipitation (ChIP) is a powerful tool to analyze such interactions in vivo.

使用染色质免疫沉淀分析c-KIT配体启动子

Multiple cellular processes, including DNA replication and repair, DNA recombination, and gene expression, require interactions between proteins and DNA. ...

Determination of the Optimal Chromosomal Location(s) for a DNA Element in Escherichia coli Using a Novel Transposon-mediated Approach

The optimal chromosomal position(s) of a given DNA element was/were determined by transposon-mediated random insertion followed by fitness selection. In bacteria, the impact of the genetic context on the function of a genetic element can be difficult to assess. Several mechanisms, including topological effects, transcriptional interference from neighboring genes, and/or replication-associated gene dosage, may affect the function of a given genetic element. Here, we describe a method that permits the random integration of a DNA element into the chromosome of Escherichia coli and select the most favorable locations using a simple growth competition experiment. The method takes advantage of a well-described transposon-based system of random insertion, coupled with a selection of the fittest clone(s) by growth advantage, a procedure that is easily adjustable to experimental needs. The nature of the fittest clone(s) can be determined by whole-genome sequencing on a complex multi-clonal population or by easy gene walking for the rapid identification of selected clones. Here, the non-coding DNA region DARS2, which controls the initiation of chromosome replication in E. coli, was used as an example. The function of DARS2 is known to be affected by replication-associated gene dosage; the closer DARS2 gets to the origin of DNA replication, the more active it becomes. DARS2 was randomly inserted into the chromosome of a DARS2-deleted strain. The resultant clones containing individual insertions were pooled and competed against one another for hundreds of generations. Finally, the fittest clones were characterized and found to contain DARS2 inserted in close proximity to the original DARS2 location.

Determination of the Optimal Chromosomal Locations for a DNA Element in Escherichia coli Using a Novel Transposon-mediated Approach

Here, the power of a transposon-mediated random insertion of a non-coding DNA element was used to resolve its optimal chromosomal position.

用新的转介导法测定大肠杆菌中 DNA 元素的最佳染色体位置 (s)

The optimal chromosomal position(s) of a given DNA element was/were determined by transposon-mediated random insertion followed by fitness selection. In ...

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

DNA replication timing is an important cellular characteristic, exhibiting significant relationships with chromatin structure, transcription, and DNA mutation rates. Changes in replication timing occur during development and in cancer, but the role replication timing plays in development and disease is not known. Zebrafish were recently established as an in vivo model system to study replication timing. Here is detailed the protocols for using the zebrafish to determine DNA replication timing. After sorting cells from embryos and adult zebrafish, high-resolution genome-wide DNA replication timing patterns can be constructed by determining changes in DNA copy number through analysis of next generation sequencing data. The zebrafish model system allows for evaluation of the replication timing changes that occur in vivo throughout development, and can also be used to assess changes in individual cell types, disease models, or mutant lines. These methods will enable studies investigating the mechanisms and determinants of replication timing establishment and maintenance during development, the role replication timing plays in mutations and tumorigenesis, and the effects of perturbing replication timing on development and disease.

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

Zebrafish were recently used as an in vivo model system to study DNA replication timing during development. Here is detailed the protocols for using zebrafish embryos to profile replication timing. This protocol can be easily adapted to study replication timing in mutants, individual cell types, disease models, and other species.

使用斑马鱼作为体内模型系统分析 DNA 复制定时

DNA replication timing is an important cellular characteristic, exhibiting significant relationships with chromatin structure, transcription, and DNA ...

CRISPR-Mediated Reorganization of Chromatin Loop Structure

Recent studies have clearly shown that long-range, three-dimensional chromatin looping interactions play a significant role in the regulation of gene expression, but whether looping is responsible for or a result of alterations in gene expression is still unknown. Until recently, how chromatin looping affects the regulation of gene activity and cellular function has been relatively ambiguous, and limitations in existing methods to manipulate these structures prevented in-depth exploration of these interactions. To resolve this uncertainty, we engineered a method for selective and reversible chromatin loop re-organization using CRISPR-dCas9 (CLOuD9). The dynamism of the CLOuD9 system has been demonstrated by successful localization of CLOuD9 constructs to target genomic loci to modulate local chromatin conformation. Importantly, the ability to reverse the induced contact and restore the endogenous chromatin conformation has also been confirmed. Modulation of gene expression with this method establishes the capacity to regulate cellular gene expression and underscores the great potential for applications of this technology in creating stable de novo chromatin loops that markedly affect gene expression in the contexts of cancer and development.

Chromatin looping plays a significant role in gene regulation; however, there have been no technological advances that allow for selective and reversible modification of chromatin loops. Here we describe a powerful system for chromatin loop re-organization using CRISPR-dCas9 (CLOuD9), demonstrated to selectively and reversibly modulate gene expression at targeted loci.

CRISPR 的染色质环结构重组

Recent studies have clearly shown that long-range, three-dimensional chromatin looping interactions play a significant role in the regulation of gene ...

Measuring Exercise Levels in Drosophila melanogaster Using the Rotating Exercise Quantification System (REQS)

Drosophila melanogaster is a new model organism for studies in exercise biology. To date, two main exercise systems, the Power Tower and the Treadwheel have been described. However, a method to measure the amount of additional animal activity induced through the exercise treatment has been lacking. The Rotating Exercise Quantification System (REQS) fills this need, providing a measure of animal activity for animals experiencing rotational exercise. This protocol details how to use the REQS to assess animal activity during rotational exercise and illustrates the type of data that can be generated. Here, we demonstrate how the REQS is used to measure sex- and strain-specific differences in exercise induced activity. The REQS can also be used to evaluate the impact of various other experimental parameters such as age, diet, or population size on exercise induced activity. In addition, it can be used to compare the efficacy of different exercise training protocols. Importantly, it provides an opportunity to standardize exercise treatments between strains, allowing the researcher to achieve equal amounts of activity between groups if needed. Thus, the REQS is a notable new resource for exercise biologists working with the Drosophila model system and complements existing exercise systems.

Measuring Exercise Levels in Drosophila melanogaster Using the Rotating Exercise Quantification System REQS

The Rotating Exercise Quantification System (REQS) can induce exercise in Drosophila melanogaster through rotation while simultaneously measuring the amount of activity performed by the animals. Here, we present a point-by-point protocol detailing how to measure activity levels of animals experiencing rotational exercise treatments using the REQS.

使用旋转运动量化系统测量果蝇中的运动水平 (满足要求)

Drosophila melanogaster is a new model organism for studies in exercise biology. To date, two main exercise systems, the Power Tower and the Treadwheel ...

Using In Vitro and In-cell SHAPE to Investigate Small Molecule Induced Pre-mRNA Structural Changes

In the process of drug development of RNA-targeting small molecules, elucidating the structural changes upon their interactions with target RNA sequences is desired. We herein provide a detailed in vitro and in-cell selective 2&#39;-hydroxyl acylation analyzed by primer extension (SHAPE) protocol to study the RNA structural change in the presence of an experimental drug for spinal muscular atrophy (SMA), survival of motor neuron (SMN)-C2, and in exon 7 of the pre-mRNA of the SMN2 gene. In in vitro SHAPE, an RNA sequence of 140 nucleotides containing SMN2 exon 7 is transcribed by T7 RNA polymerase, folded in the presence of SMN-C2, and subsequently modified by a mild 2&#39;-OH acylation reagent, 2-methylnicotinic acid imidazolide (NAI). This 2&#39;-OH-NAI adduct is further probed by a 32P-labeled primer extension and resolved by polyacrylamide gel electrophoresis (PAGE). Conversely, 2&#39;-OH acylation in in-cell SHAPE takes place in situ with SMN-C2 bound cellular RNA in living cells. The pre-mRNA sequence of exon 7 in the SMN2 gene, along with SHAPE-induced mutations in the primer extension, was then amplified by PCR and subject to next-generation sequencing. Comparing the two methodologies, in vitro SHAPE is a more cost-effective method and does not require computational power to visualize results. However, the in vitro SHAPE-derived RNA model sometimes deviates from the secondary structure in a cellular context, likely due to the loss of all interactions with RNA-binding proteins. In-cell SHAPE does not need a radioactive material workplace and yields a more accurate RNA secondary structure in the cellular context. Furthermore, in-cell SHAPE is usually applicable for a larger range of RNA sequences (~1,000 nucleotides) by utilizing next-generation sequencing, compared to in vitro SHAPE (~200 nucleotides) that usually relies on PAGE analysis. In case of exon 7 in SMN2 pre-mRNA, the in vitro and in-cell SHAPE derived RNA models are similar to each other.

Detailed protocols for both in vitro and in-cell selective 2&#39;-hydroxyl acylation analyzed by primer extension (SHAPE) experiments to determine the secondary structure of pre-mRNA sequences of interest in the presence of an RNA-targeting small molecule are presented in this article.

利用体外和细胞内形状研究小分子诱导的预 mrna 结构变化

In the process of drug development of RNA-targeting small molecules, elucidating the structural changes upon their interactions with target RNA sequences ...

Determining the Egg Fertilization Rate of Bemisia tabaci Using a Cytogenetic Technique

A few species of sap-sucking whiteflies are some of the most damaging terrestrial pests worldwide because of the crop damage they inflict and plant viruses they vector. Despite numerous studies of the biology of these species in different environments, a key life history parameter, offspring sex ratios, has received little attention, yet is important for predicting population dynamics. The primary sex ratio (sex ratio at oviposition) of Bemisia tabaci has never been reported but can be found by determining the egg fertilization rate of this haplodiploid insect. The technique involves the dechorionation of eggs with bleach, a series of fixation steps, and the application of the general DNA fluorescent stain, DAPI (4&#8242;,6-diamidino-2-phenylindole, a DNA-binding fluorescent dye), to bind to female and male pronuclei. Here, we present the technique, and an example of its application, to test whether an endosymbiotic bacterium, Rickettsia sp. nr. bellii, influenced the primary sex ratio of B. tabaci. This method may assist in population studies of whiteflies, or in determining if sex allocation exists with certain environmental stimuli.

Determining the Egg Fertilization Rate of Bemisia tabaci Using a Cytogenetic Technique

We present a simple cytogenetic technique using 4&#8242;,6-diamidino-2-phenylindole (DAPI) to determine the fertilization rate and primary sex ratio of the haplodiploid invasive pest Bemisia tabaci.

用细胞遗传学技术测定芽孢杆菌的受精率

A few species of sap-sucking whiteflies are some of the most damaging terrestrial pests worldwide because of the crop damage they inflict and plant ...

Characterization of In Vitro Differentiation of Human Primary Keratinocytes by RNA-Seq Analysis

Human primary keratinocytes are often used as in vitro models for studies on epidermal differentiation and related diseases. Methods have been reported for in vitro differentiation of keratinocytes cultured in two-dimensional (2D) submerged manners using various induction conditions. Described here is a procedure for 2D in vitro keratinocyte differentiation method by contact inhibition and subsequent molecular characterization by RNA-seq. In brief, keratinocytes are grown in defined keratinocyte medium supplemented with growth factors until they are fully confluent. Differentiation is induced by close contacts between the keratinocytes and further stimulated by excluding growth factors in the medium. Using RNA-seq analyses, it is shown that both 1) differentiated keratinocytes exhibit distinct molecular signatures during differentiation and 2) the dynamic gene expression pattern largely resembles cells during epidermal stratification. As for comparison to normal keratinocyte differentiation, keratinocytes carrying mutations of the transcription factor p63 exhibit altered morphology and molecular signatures, consistent with their differentiation defects. In conclusion, this protocol details the steps for 2D in vitro keratinocyte differentiation and its molecular characterization, with an emphasis on bioinformatic analysis of RNA-seq data. Because RNA extraction and RNA-seq procedures have been well-documented, it is not the focus of this protocol. The experimental procedure of in vitro keratinocyte differentiation and bioinformatic analysis pipeline can be used to study molecular events during epidermal differentiation in healthy and diseased keratinocytes.

Presented here is a stepwise procedure for in vitro differentiation of human primary keratinocytes by contact inhibition followed by characterization at the molecular level by RNA-seq analysis.

通过RNA-Seq分析，人类原主角细胞的体外分化特征

Human primary keratinocytes are often used as in vitro models for studies on epidermal differentiation and related diseases. Methods have been reported ...