The authors would like to acknowledge the contribution of our colleagues Sandrine Roch-Lefevre and Eric Gregoire of the Institute of Radioprotection and Nuclear Safety (Paris, France). This work was supported by the Government of Canada Science and Technology program at Canadian Nuclear Laboratories (Chalk River, Ontario, Canada), the CANDU Owners Group (Toronto, Ontario, Canada), the Canadian Nuclear Safety Commission and by the Institute of Radioprotection and Nuclear Safety (Paris, France).

所有的鼠标操作和处理程序应遵循规定由立法机关和/或动物保健计划，并批准由当地动物保护委员会的规则。在这个协议中描述的所有方法均按照加拿大理事会关于动物保护与当地动物保护委员会批准的指导进行。 注意：有放射性的所有工作（包括但不限于处理氚，γ外照射，放射性处理的动物组织，床上用品废物）要坚持规定由立法机关和/或辐射防护部门的规则，并授权执行人员经过认证​​的实验室和/或设施。 1.动物工作<ol><li>动物分组<ol><li>如果小鼠从外部组织到达，适应环境的动物在动物设施至少10天。如果研究包括多个治疗组，保证了动物的供应商可以发送人升动物单批。 </li><li>随机分配小鼠到10只小鼠每组的治疗组。如果使用雄性小鼠C57BL / 6株，有动物的供应商预先安排的非常年轻的C57BL / 6雄性随机在他们的设施，以缓解侵略的问题。 </li></ol></li><li>慢性暴露小鼠对内部β射线<ol><li>通过稀释HTO和OBT溶液（氚化脯氨酸，丙氨酸和甘氨酸的氨基酸的混合物）的原始库存制备氚在小鼠的饮用水工作浓度（10 kBq / L和1活度/ L）。 </li><li>通过用液体闪烁计数器测量放射性验证在饮用水的最终浓度的氚。调整浓度，如果需要的话。 </li><li>通过提供自由采食的氚水对待动物一个月。如果处理水在两周内变低，多补充水分。否则，两周改变瓶。 </li><li>对待假照控制小鼠相同。让他们在一个单独的“干净”的房间或在正气压以避免氚交叉污染从空气中一个单独的“干净”的架子。 </li></ol></li><li>慢性暴露小鼠对外部γ射线 注意：γ-辐照装置/设备将根据位置而变化。 </li><li>使用剂量的可利用的方法，确定从γ射线源，其具有产生所要求的剂量率的辐射场的距离。确保涵盖了动物笼舍的范围内辐射场的均匀性。 注意：1.7μGy/小时相当于1活度/在物L氚产生的剂量率。 </li><li>通过将鼠标笼在一个月步骤1.3.1确定的距离暴露的小鼠。减少γ射线的每日中断30分钟。用热释光剂量计来测量总吸收剂量。 </li></ol> 2.牺牲和采样<o升&gt; <li>制备无菌手术器械，60毫米培养皿具有插入70微米的细胞过滤网，15毫升和1.5毫升管，所有这些都在生物安全柜补充有5％胎牛血清（FBS），并置于RPMI培养基。确保工作的无菌性是至关重要的。 </li><li>分配5毫升RPMI培养基的成各60 10mm培养皿中</li><li>牺牲采用经当地动物保护委员会的方法小鼠。颈椎脱位是在这个协议中描述的脾细胞工作的一个不错的选择。然而，如果需要的血液样本进行化验等（ 例如，对于渔业部的松弛素微块或其他常见的遗传毒性试验），麻醉与异氟醚后使用心内穿刺。 </li><li>将动物背卧，用头从你背向。喷小鼠用70％的乙醇。使用镊子，前方抢皮肤到尿道口，使一个小切口在会阴区域剪刀。 <o升&gt; <li>从这个切口，沿腹正中线向胸腔切开，小心，只切开皮肤，而不是肌壁下方。解剖皮肤远离中线。 </li><li>把握腹壁用钳子和沿肌肉壁打开腹腔的中间轴线切割。 </li></ol></li><li>轻轻夹住它，用无菌镊子轻轻拉动它同时切掉用剪刀结缔组织切除脾脏。 </li><li>剪一小块脾（大约1/10脾），地点在1.5毫升管。卡扣冷冻在液氮中用于随后存储在-80℃下进行补充试验。 </li><li>放置脾的其余成60毫米陪替氏培养皿内的细胞过滤用5ml预分配的RPMI介质。 </li><li>继续下一个鼠标。 注意：提取脾脏，可以保持60毫米培养皿与媒体（长达2小时），而小鼠的其余部分是sacrificED。取决于参与，第5和15只小鼠之间的人数可以在一天之内进行处理。 </li></ol> 3.准备脾细胞培养的<ol><li>通过切碎细胞过滤用无菌镊子一弯曲端内均匀脾。 </li><li>除去细胞过滤并收集从60mm培养皿的过滤细胞悬液到15毫升管。 </li><li>冲洗细胞过滤用5ml介质收集所述细胞的剩余部分。 </li><li>离心管，在300×g离心5分钟，在RT。 </li><li>倒出上清液并重新悬浮沉淀在10ml的RPMI。 </li><li>离心管，在300×g离心5分钟，在RT。 </li><li>倒出上清液并重新悬浮沉淀在5毫升的RPMI。 </li><li>传输4 ml的细胞悬浮液加入25毫升组织培养烧瓶中，并转移到CO 2培养箱（37℃，5％CO 2，80％湿度） </li></ol> 4.挑战照射和修复<ol><li>转移剩下的1 ml的细胞悬浮液从步骤3.8到1.5毫升管在冰上和离心机在300×g离心5分钟，在4℃。此样品中测量DNA损伤将代表两个治疗诱导损伤和t = 0的数据点的DNA修复曲线的构造。 </li><li>小心吸用真空泵上清液。轻轻在1ml TBS缓冲液（20mM的Tris pH值7.4，150 mM氯化钠，2.7毫摩尔KCl）中重新悬浮沉淀。 </li><li>离心管，在300×g离心5分钟，在4℃。小心吸用真空泵上清液。轻轻在300微升TBS的重新悬浮颗粒。 </li><li>在混合的同时在涡流在低速，加入700μl-20℃的100％乙醇。盖上盖子，倒转几次混合。 </li><li>储存的样品在-20℃。脾细胞固定在乙醇可以储存在-20℃至少12个月没有在γH2AX信号中的任何明显的损失。 </li><li>照射步骤3.8的脾细胞培养与一个具有挑战性的γ-RAD<html> 2戈瑞iation剂量在使用可用照射装置的剂量率≥200毫戈瑞/分。 注意：该照射的目的是诱导的DNA DSB挑战修复机制。 X射线，也可以用于此目的。 </li><li>立即返回培养到CO 2培养箱。 </li><li>具有挑战性的2戈瑞照射后1小时，取出从孵化器文化的一个生物安全柜。收集有代表性的样品等分试样，轻轻重新悬浮使用移液器和细胞悬浮液转移1ml至1.5ml试管在冰上的细胞。返回的细胞培养在CO 2培养箱中培养。 </li><li>离心管，在300×g离心5分钟，在4℃。小心吸用真空泵上清液。轻轻在1ml TBS中重新悬浮沉淀。 </li><li>重复步骤4.3 - 4.5。 </li><li> 24小时的具有挑战性的2 Gy的γ射线照射，收获后按照步骤4.8中所述的= 24小时采样修复 - 4.10。 </li></ol>TLE“&gt; 5。免疫荧光标记与抗γH2AX抗体注意：通常情况下，15 - 每天30个样品，可免疫荧光标记的和由在单个运行流式细胞仪测定。以确保治疗组之间的精确比较，包括从每组样品（多个）。请参阅参考16的进一步细节。 <ol><li>制备管的样本数的在冰上的标记集进行处理和地点。使用12×75mm的无上限的玻璃管。不需要这个工作无菌。包括与二级抗体标记只有一个额外的历史对照样品（“第二只抗体”）。使用此历史对照样品中每一个流式细胞仪的一项研究中运行。 </li><li>加入0.5毫升冰冷的TBS中至每个管。 </li><li>为-20℃，涡流除去固定的脾细胞5秒和0.5毫升等分用TBS转移到准备好的试管中。将样品的剩余部分返回到-20℃的储存和保持作为备用样品。 </li><li>离心机的脾细胞在300×g离心5分钟，在4℃。滗上清液轻轻在1ml冰冷的TBS中含1％FBS的重悬浮沉淀</li><li>离心细胞，在300×g离心5分钟，在4℃下。滗析出上清液并轻轻在1ml TST缓冲液（0.05％的Triton X-100，在TBS 2％FBS）中重新悬浮沉淀。孵育在冰上20分钟。 </li><li>离心细胞，在300×g离心5分钟，在4℃下。滗析出上清液并轻轻在200μl初级抗γH2AX抗体的稀释1重新悬浮沉淀：150中TST缓冲器。用200微升TST为“第二只AB”对照样品。 </li><li>在旋转振荡器上在45°角位置管 - 60°和摇1.5小时，在300×g离心在RT。 </li><li>而管温育，准备在一个1缀合有Alexa-488山羊抗小鼠二级抗体的足够体积（200微升/样品）：200稀释在TST缓冲器。 注：所有涉及的Alexa-488的工作缀合抗体（步骤5.10至5.16以下）应该光线昏暗的条件下完成，并且标记的样品应当通过包装管在铝箔避光。 </li><li>一旦1.5小时温育完成后，加入1毫升的冰冷的TBS含有2％FBS的每个试管和离心机在300×g离心5分钟，在4℃。滗析出上清液并轻轻于1ml含有2％FBS的TBS中重新悬浮沉淀。 </li><li>离心机在300×g离心5分钟，在4℃。滗上清液轻轻从步骤5.8重悬沉淀在200μl第二抗体溶液。 </li><li>孵育1小时的摇动平台上的样品如在步骤5.7。 </li><li>加入1毫升的冰冷的TBS中含1％FBS中。 </li><li>离心机在300×g离心5分钟，在4℃。滗上清液轻轻在1ml冰冷的TBS重悬沉淀。 </li><li>离心机在300×g离心5分钟，在4℃。滗析出上清液并轻轻在0.5含有50微克/毫升的TBS毫升重新悬浮沉淀碘化。 </li><li>孵育5分钟，在室温避光。 </li><li>在流式细胞仪上使用仪器专用协议16分析的样品。读每个样品至少10,000个细胞。 </li></ol>

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J. <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+stress+genes+by+low+doses+of+gamma+rays.">Induction of stress genes by low doses of gamma rays.</a> Radiat Res. 152 (3), 225-231 (1999).</li></ol>

The authors declare that there are no known competing financial interests.

本文提出的协议是用于进行大规模的老鼠体内研究审查低水平各种化学和物理因素，包括电离辐射的遗传毒性作用是有用的。我们独特的无特定病原体动物设施配备了150 GammaBeam辐射和一个30米长的照射大厅允许进行涉及低或非常低剂量率照射在数百甚至数千老鼠终身学习。较小的辐照设施慢性低剂量照射，可设置在一所大学或医院的环境，放射生物学的研究进行例行和辐射防护指导方针/法...

显著争论低或非常低剂量的电离辐射和公众的恐惧辐射，由广岛和长崎原子弹爆炸和珍稀核电站事故的图像驱动的潜在有害影响（通过大众媒体加剧），导致了很严格的辐射防护监管和有潜在不能科学合理的标准。在过去的三十年中，许多报告已经证明了两者的不足和有害诱导低剂量辐射1-4潜在有益的生物效应的存在。主要辐射的健康危险因素是癌症的概率，根据对原子弹爆炸幸存者接受高或中等剂量的辐射流行病学研究估计。这些数据（所谓的线性无阈值或LNT模型）的线性外推来估算的癌症风险在低剂量。然而，这种方法并没有收到世界通用的科学验收并被大量辩论5。 很明显，更多的研究来澄清这一问题，并可能提高辐射防护标准。这些研究应当包括（最好为外推效果对人类）和这样的最终点作为DNA损伤率，DNA修复和诱变慢性治疗（环境和职业暴露最佳逼近）， 在体内动物模型。已知的是，DNA的是用于破坏性辐射效应和不完整或错误修复可导致诱变和癌发展6的主要目标。 DNA双链断裂（DSB）是最有害类型的DNA损伤之一，并且可能会导致细胞死亡和肿瘤发生7。据，因此，重要的是能够可靠地和精确地测量DSB暴露于低剂量辐射和/或其他压力，如化学污染物后的水平。其中最敏感和特异的标记物的DNA DSB的是磷酸化组蛋白H2AX，称为γH2AX8，但其他标记物和方法已被建议9,10。据估计，数千H2AX分子，在诱导的DSB的附近，是参与γH2AX的形成可进行个体的DSB的通过免疫荧光标记的检测与抗γH2AX抗体和荧光显微镜11。该反应是非常快的，分30至60达到其最大值。有证据表明γH2AX利于修复DNA DSB通过吸引其他修复因子来休息的场所，并通过改变染色质结构来断锚定DNA末端，并提供访问其他修复蛋白（12综述）。在修复DNA DSB的完成，得到γH2AX去磷酸化和/或发生退化，新合成的分子H2AXγH2AX替代染色质10的受灾地区。监测γH2AX的形成和损失，因此，提供的DNA DSB修复动力学的精确估计。这种方法已被用于研究修复DSB在照射高剂量的辐射和其速率和残留的DSB水平的各种人肿瘤细胞系已被证实与放射敏感性13-15。 我们修改了这个实验方法并将其应用到小鼠体内研究，探讨低剂量慢性γ-和β辐射对DNA DSB水平和维修（图1）的影响。首先，我们展示了一个方法来执行的小鼠的长期慢性暴露于β辐射由氚或者发射（氢-3）中氚化水（HTO）的形式或作为有机结合的氚（OBT）溶解在饮用水。这两种形式都预期累积和/或分发不同的机构，因此，产生不同的生物学效应。这两种形式是核工业的潜在危险。这种治疗方法由长期暴露于γ射线在等效剂量率并联以允许正确比较的两个辐射类型，这对于它们的相对生物学有效性的评价是至关重要的。的β-辐射是由电子的，使得它从γ射线，高能光子非常不同。由于这种差异，Β辐射表示主要是内部的健康危害和可能产生较γ射线不同的生物学效应。这种并发症导致曝光过度的监管，β射线通过HTO发出显著争议。因此，HTO在饮用水的公共管理水平各不相同，从100贝可/ L的欧洲至75,000贝可/ L，在澳大利亚。它是，因此，重要的是要比较HTO生物效应等效剂量γ射线的。其次，DNA DSB的速率是使用免疫荧光标记的γH2AX检测完成慢性暴露后测定分离的脾细胞通过流式细胞仪主编。这允许通过在体内暴露造成DNA损伤的程度的评价。然而，这是明智的预期，这些低水平暴露可能不会产生DNA DSB任何检出率;相反，一些隐藏更改/应答可以预期这将影响到细胞的修复DNA损伤的能力。这些变化中，如果发现，可以是刺激（产生有益的影响）或抑制（产生有害的影响）。该协议允许通过用高剂量的辐射，产生一个显著量损坏的挑战所提取的脾细胞揭示这样的变化（ 例如 ，2戈瑞产生每细胞约50的DNA DSB或3 - 5倍，增加总的γH2AX水平） 。接着，形成与γH2AX的损失，这反映了DSB修复的起始和结束时，通过流式细胞术监测。以这种方式，不仅基底和DNA DSB的治疗引起的水平可以被测量，但还对细胞的能力的潜在影响作出回​​应和修复DNA损伤诱导的高得多的水平应激。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>HTO: tritiated water, [3H]</td>
			<td>locally obtained from a nuclear reactor&#160;</td>
			<td></td>
			<td>stock activity 3.7 GBq/mL;&#160; can be substituted with HTO from Perkin Elmer</td>
		</tr>
		<tr>
			<td>OBT: Alanine, L-[3-3H]: organically bound tritium (OBT)</td>
			<td>Perkin Elmer</td>
			<td>NET348005MC</td>
			<td>1mCi/mL (185 MBq)</td>
		</tr>
		<tr>
			<td>OBT: Glycine, [2-3H]: organically bound tritium</td>
			<td>Perkin Elmer</td>
			<td>NET004005MC</td>
			<td>1mCi/mL (185 MBq)</td>
		</tr>
		<tr>
			<td>OBT: Proline, L-[2,3-3H]: organically bound tritium (OBT)</td>
			<td>Perkin Elmer</td>
			<td>NET323005MC</td>
			<td>1mCi/mL (185 MBq)</td>
		</tr>
		<tr>
			<td>tritiated water, [3H] (HTO)</td>
			<td>Perkin Elmer</td>
			<td>NET001B005MC</td>
			<td>substitute for HTO of local origin&#160;&#160;</td>
		</tr>
		<tr>
			<td>GammaBeam 150 irradiator</td>
			<td>Atomic Energy of Canada Limited</td>
			<td>locally manufactured</td>
			<td>can be substituted with another g-radiation source of sufficiently low activity&#160;</td>
		</tr>
		<tr>
			<td>tween-20</td>
			<td>Sigma Aldrich</td>
			<td>P1379-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI</td>
			<td>Fisher Scientific</td>
			<td>SH3025501</td>
			<td>Hyclone RPMI 1640 with L-Glutamine and HEPES 500mL</td>
		</tr>
		<tr>
			<td>fetal bovine serum</td>
			<td>Sigma Aldrich</td>
			<td>F1051-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-gH2AX antibody, clone JBW301</td>
			<td>Millipore</td>
			<td>05-636</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa fluor-488 goat anti-mouse antibody</td>
			<td>Life Technologies (formerly Invitrogen)</td>
			<td>A21121</td>
			<td></td>
		</tr>
		<tr>
			<td>propidium iodine</td>
			<td>Sigma Aldrich</td>
			<td>P4864-10ML</td>
			<td>1mg/mL</td>
		</tr>
		<tr>
			<td>ethanol</td>
			<td>Commercial Alcohols</td>
			<td>P006-EAAN</td>
			<td>500ml bottles Absolute Ethanol</td>
		</tr>
		<tr>
			<td>1.5 mL tubes</td>
			<td>Fisher Scientific</td>
			<td>2682550</td>
			<td>microcentrifuge tubes</td>
		</tr>
		<tr>
			<td>15 mL tubes</td>
			<td>Fisher Scientific</td>
			<td>05-539-5</td>
			<td>sterile polypropylene centrifuge tubes</td>
		</tr>
		<tr>
			<td>liquid nitrogen</td>
			<td>Linde</td>
			<td>P110403</td>
			<td></td>
		</tr>
		<tr>
			<td>12 x 75 mm mL uncapped glass tubes</td>
			<td>Fisher Scientific</td>
			<td>K60B1496126</td>
			<td>disposable borosilicate glass tubes with plain end</td>
		</tr>
		<tr>
			<td>T25 flasks</td>
			<td>VWR</td>
			<td>CA15708-120</td>
			<td>nunc tisue culture 25ml flask (supplier no. 156340)</td>
		</tr>
		<tr>
			<td>scissors</td>
			<td>Fine Science Tools</td>
			<td>14068-12</td>
			<td>Wagner scissors 12cm sharp/sharp</td>
		</tr>
		<tr>
			<td>forceps, straight</td>
			<td>Fine Science Tools</td>
			<td>11008-13</td>
			<td>Semken forceps 13cm, straight</td>
		</tr>
		<tr>
			<td>forceps, curved</td>
			<td>Fine Science Tools</td>
			<td>11003-12</td>
			<td>Narrow Pattern forceps 12 cm, curved</td>
		</tr>
		<tr>
			<td>60 mm petri dishes</td>
			<td>VWR</td>
			<td>CA25382-100</td>
			<td>BD Falcon tissue culture dish 60x15mm</td>
		</tr>
		<tr>
			<td>cell strainers, 70 mm</td>
			<td>Fisher Scientific</td>
			<td>08-771-2</td>
			<td>Falcon cell strainers 50/case</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PBS recipe: 1 tablet dissolved in 200mL of deionized water, adjust pH to 7.4 if needed.</td>
			<td>Sigma Aldrich</td>
			<td>P4417-100TAB</td>
			<td>Phosphate Buffered Saline Tablets</td>
		</tr>
		<tr>
			<td>TBS recipe: to make 10X Stock,&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>30g TRIS HCl</td>
			<td>Sigma Aldrich</td>
			<td>T3253-1KG</td>
			<td>Trizma hydrochloride</td>
		</tr>
		<tr>
			<td>88g NaCl</td>
			<td>Fisher Scientific</td>
			<td>S271-500</td>
			<td>Sodium Chloride</td>
		</tr>
		<tr>
			<td>2g KCl</td>
			<td>Fisher Scientific</td>
			<td>P217-500</td>
			<td>Potassium Chloride</td>
		</tr>
		<tr>
			<td>Dissolve in 1L of deionized water, adjust pH to 7.4</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

measuring dna damage and repair in mouse splenocytes after chronic in vivo exposure to very low doses of beta- and gamma-radiation

低剂量的辐射暴露可能产生多种生物效应是在由高辐射剂量所产生的影响的数量和质量不同。处理适当和有科学依据的方式与环境，职业健康和公共卫生安全问题在很大程度上依赖于准确测量低剂量的污染物，如电离辐射，化学物质的生物效应的能力。 DNA损伤和修复是健康风险最重要的早期指标，由于其潜在的长期后果，例如癌症。在这里，我们描述了一种协议来研究慢性体内暴露于低剂量的γ-和β射线对DNA损伤和修复在小鼠脾细胞中的作用。采用DNA双链断裂的一个普遍接受的标记，磷酸化组蛋白H2AX被称为γH2AX，我们展示它如何被用于评估DNA损伤的不仅是水平，而且还改变在DNA修复能力可能通过体内暴露低剂量制备。流式细胞仪允许在大量样品的免疫荧光标记的γH2AX的快速，准确和可靠的测量。 DNA双链断裂修复可以通过暴露提取脾细胞的2戈瑞一个具有挑战性的剂量以产生足够数量的DNA的破坏触发修理和通过测量诱导（1小时后照射）和残留的DNA损伤进行评估（24小时照射后）。残余DNA损伤将指示不完全修复和长期的基因组不稳定和癌症的风险。结合其它测定法和最终点，可以很容易地在这种被测定体内研究（ 例如 ，染色体畸变，在骨髓的网织红细胞微核频率，基因表达等），这种方法允许的生物效应的准确和上下文评价低水平压力。

图2示出流的例子术预期脾图形使用此处描述的方法制备。根据&lt;电子音量侧散射&gt;散点图细胞被第一选通（ 图2A和2D;电子体积相当于前向散射）。 FL3 /碘化丙直方图（ 图2B和2E）确认正常细胞周期分布。意味着使用FL1通道计算γH2AX信号证实了&gt; 2倍的增加的DNA DSB在2-戈瑞照射细胞（ 图2F）相比于未处理的对照（ ?...

Watch this Scientific Journal Video about Measuring DNA Damage and Repair in Mouse Splenocytes After Chronic In Vivo Exposure to Very Low Doses of Beta- and Gamma-Radiation at JoVE.com

Measuring DNA Damage and Repair in Mouse Splenocytes After Chronic In Vivo Exposure to Very Low Doses of Beta- and Gamma-Radiation

A protocol to evaluate changes in DNA damage levels and DNA repair capacity that may be induced by chronic in vivo low dose irradiation in mouse spleen lymphocytes, by measuring phosphorylated histone H2AX, a marker of DNA double-strand breaks, using flow cytometry is presented.

测量DNA损伤与修复小鼠脾细胞慢性后<em&gt;在体内</em&gt;暴露于β和γ辐射剂量很低

Low dose radiation exposure may produce a variety of biological effects that are different in quantity and quality from the effects produced by high ...

measuring-dna-damage-repair-mouse-splenocytes-after-chronic-vivo

Research

JoVE Journal

Biology

Measuring DNA Damage and Repair in Mouse Splenocytes After Chronic In Vivo Exposure to Very Low Doses of Beta- and Gamma-Radiation

11.0K 次观看.  Canadian Nuclear Laboratories. A protocol to evaluate changes in DNA damage levels and DNA repair capacity that may be induced by chronic in vivo low dose irradiation in mouse spleen lymphocytes, by measuring phosphorylated histone H2AX, a marker of DNA double-strand breaks, using flow cytometry is presented. 

视频: Measuring DNA Damage and Repair in Mouse Splenocytes After Chronic In Vivo Exposure to Very Low Doses of Beta- and Gamma-Radiation

Imaging Mismatch Repair and Cellular Responses to DNA Damage in Bacillus subtilis

Both prokaryotes and eukaryotes respond to DNA damage through a complex set of physiological changes. Alterations in gene expression, the redistribution of existing proteins, and the assembly of new protein complexes can be stimulated by a variety of DNA lesions and mismatched DNA base pairs. Fluorescence microscopy has been used as a powerful experimental tool for visualizing and quantifying these and other responses to DNA lesions and to monitor DNA replication status within the complex subcellular architecture of a living cell. Translational fusions between fluorescent reporter proteins and components of the DNA replication and repair machinery have been used to determine the cues that target DNA repair proteins to their cognate lesions in vivo and to understand how these proteins are organized within bacterial cells. In addition, transcriptional and translational fusions linked to DNA damage inducible promoters have revealed which cells within a population have activated genotoxic stress responses. In this review, we provide a detailed protocol for using fluorescence microscopy to image the assembly of DNA repair and DNA replication complexes in single bacterial cells. In particular, this work focuses on imaging mismatch repair proteins, homologous recombination, DNA replication and an SOS-inducible protein in Bacillus subtilis. All of the procedures described here are easily amenable for imaging protein complexes in a variety of bacterial species.

Imaging Mismatch Repair and Cellular Responses to DNA Damage in Bacillus subtilis

A detailed protocol is described for imaging the real time formation of DNA repair complexes in Bacillus subtilis cells.

成像错配修复和DNA损伤的细胞反应枯草芽孢杆菌</em

Both prokaryotes and eukaryotes respond to DNA damage through a complex set of physiological changes. Alterations in gene expression, the redistribution ...

科研

生物学

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

活体成像和治疗卵巢癌的原位小鼠模型

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

Visualization of MG53-mediated Cell Membrane Repair Using in vivo and in vitro Systems

Repair of acute injury to the cell membrane is an elemental process of normal cellular physiology, and defective membrane repair has been linked to many degenerative human diseases. The recent discovery of MG53 as a key component of the membrane resealing machinery allows for a better molecular understanding of the basic biology of tissue repair, as well as for potential translational applications in regenerative medicine. Here we detail the experimental protocols for exploring the in vivo function of MG53 in repair of muscle injury using treadmill exercise protocols on mouse models, for testing the ex vivo membrane repair capacity by measuring dye entry into isolated muscle fibers, and for monitoring the dynamic process of MG53-mediated vesicle trafficking and cell membrane repair in cultured cells using live cell confocal microscopy.

Visualization of MG53-mediated Cell Membrane Repair Using in vivo and in vitro Systems

Described here are protocols used to visualize the dynamic process of MG53-mediated cell membrane repair in whole animals and at the cellular level. These methods can be applied to investigate the cell biology of plasma membrane resealing and regenerative medicine.

MG53介导的细胞膜使用维修的可视化在体内在体外系统

Repair of acute injury to the cell membrane is an elemental process of normal cellular physiology, and defective membrane repair has been linked to many ...

gDNA Enrichment by a Transposase-based Technology for NGS Analysis of the Whole Sequence of BRCA1, BRCA2, and 9 Genes Involved in DNA Damage Repair

The widespread use of Next Generation Sequencing has opened up new avenues for cancer research and diagnosis. NGS will bring huge amounts of new data on cancer, and especially cancer genetics. Current knowledge and future discoveries will make it necessary to study a huge number of genes that could be involved in a genetic predisposition to cancer. In this regard, we developed a Nextera design to study 11 complete genes involved in DNA damage repair. This protocol was developed to safely study 11 genes (ATM, BARD1, BRCA1, BRCA2, BRIP1, CHEK2, PALB2, RAD50, RAD51C, RAD80, and TP53) from promoter to 3&#39;-UTR in 24 patients simultaneously. This protocol, based on transposase technology and gDNA enrichment, gives a great advantage in terms of time for the genetic diagnosis thanks to sample multiplexing. This protocol can be safely used with blood gDNA.

gDNA Enrichment by a Transposase-based Technology for NGS Analysis of the Whole Sequence of BRCA1, BRCA2, and 9 Genes Involved in DNA Damage Repair

gDNA enrichment for NGS sequencing is an easy and powerful tool for the study of constitutional mutations. In this article, we present the procedure to analyse simply the complete sequence of 11 genes involved in DNA damage repair.

基因组DNA富集转座为基础的技术的整个序列的农工商分析<em&gt; BRCA1基因</em&gt;<em&gt; BRCA2</em&gt;和9个基因参与DNA损伤修复

The widespread use of Next Generation Sequencing has opened up new avenues for cancer research and diagnosis. NGS will bring huge amounts of new data on ...

A Highly Reproducible and Straightforward Method to Perform In Vivo Ocular Enucleation in the Mouse after Eye Opening

Enucleation or the surgical removal of an eye can generally be considered as a model for nerve deafferentation. It provides a valuable tool to study the different aspects of visual, cross-modal and developmental plasticity along the mammalian visual system1-4.
Here, we demonstrate an elegant and straightforward technique for the removal of one or both eyes in the mouse, which is validated in mice of 20 days old up to adults. Briefly, a disinfected curved forceps is used to clamp the optic nerve behind the eye. Subsequently, circular movements are performed to constrict the optic nerve and remove the eyeball. The advantages of this technique are high reproducibility, minimal to no bleeding, rapid post-operative recovery and a very low learning threshold for the experimenter. Hence, a large amount of animals can be manipulated and processed with minimal amount of effort. The nature of the technique may induce slight damage to the retina during the procedure. This side effect makes this method less suitable as compared to Mahajan et al. (2011)5 if the goal is to collect and analyze retinal tissue. Also, our method is limited to post-eye opening ages (mouse: P10 - 13 onwards) since the eyeball needs to be displaced from the socket without removing the eyelids. The in vivo enucleation technique described in this manuscript has recently been successfully applied with minor modifications in rats and appears useful to study the afferent visual pathway of rodents in general.

A Highly Reproducible and Straightforward Method to Perform In Vivo Ocular Enucleation in the Mouse after Eye Opening

The removal of eyes, also called enucleation, provides a useful strategy to study aspects of visual, cross-modal, and developmental plasticity along the mammalian visual system since it induces irreversible partial (monocular) or complete (binocular) vision loss. Here we describe a highly reproducible and straightforward approach to perform in vivo enucleation.

一个高度可重复的和直接的方法来执行<em&gt;体内</em&gt;眼剜除术中的鼠标眼睛开放后

Enucleation or the surgical removal of an eye can generally be considered as a model for nerve deafferentation. It provides a valuable tool to study the ...

Measuring Glutathione-induced Feeding Response in Hydra

Hydra is among the most primitive organisms possessing a nervous system and chemosensation for detecting reduced glutathione (GSH) for capturing the prey. The movement of prey organisms causes mechanosensory discharge of the stinging cells called nematocysts from hydra, which are inserted into the prey. The feeding response in hydra, which includes curling of the tentacles to bring the prey towards the mouth, opening of the mouth and consequent engulfing of the prey, is triggered by GSH present in the fluid released from the injured prey. To be able to identify the molecular mechanism of the feeding response in hydra which is unknown to date, it is necessary to establish an assay to measure the feeding response. Here, we describe a simple method for the quantitation of the feeding response in which the distance between the apical end of the tentacle and mouth of hydra is measured and the ratio of such distance before and after the addition of GSH is determined. The ratio, called the relative tentacle spread, was found to give a measure of the feeding response. This assay was validated using a starvation model in which starved hydra show an enhanced feeding response in comparison with daily fed hydra.

Here we describe a simple assay for the quantification of the feeding response in hydra induced by the reduced form of glutathione. This assay relies on measuring the distance between the apical end of the tentacle and mouth of hydra.

测量水润谷胱甘肽诱导饲养响应

Hydra is among the most primitive organisms possessing a nervous system and chemosensation for detecting reduced glutathione (GSH) for capturing the prey. ...

Detection and Analysis of DNA Damage in Mouse Skeletal Muscle In Situ Using the TUNEL Method

Terminal deoxynucleotidyl transferase (TdT) deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) is the method of using the TdT enzyme to covalently attach a tagged form of dUTP to 3&#8217; ends of double- and single-stranded DNA breaks in cells. It is a reliable and useful method to detect DNA damage and cell death in situ. This video describes dissection, tissue processing, sectioning, and fluorescence-based TUNEL labeling of mouse skeletal muscle. It also describes a method of semi-automated TUNEL signal quantitation. Inherent normal tissue features and tissue processing conditions affect the ability of the TdT enzyme to efficiently label DNA. Tissue processing may also add undesirable autofluorescence that will interfere with TUNEL signal detection. Therefore, it is important to empirically determine tissue processing and TUNEL labeling methods that will yield the optimal signal-to-noise ratio for subsequent quantitation. The fluorescence-based assay described here provides a way to exclude autofluorescent signal by digital channel subtraction. The TUNEL assay, used with appropriate tissue processing techniques and controls, is a relatively fast, reproducible, quantitative method for detecting apoptosis in tissue. It can be used to confirm DNA damage and apoptosis as pathological mechanisms, to identify affected cell types, and to assess the efficacy of therapeutic treatments in vivo.

Detection and Analysis of DNA Damage in Mouse Skeletal Muscle In Situ Using the TUNEL Method

This video describes dissection, tissue processing, sectioning, and fluorescence-based TUNEL labeling of mouse skeletal muscle. It also describes a method for semi-automated analysis of TUNEL labeling.

检测和DNA损伤分析小鼠骨骼肌<em&gt;原位</em&gt;使用TUNEL法

Terminal deoxynucleotidyl transferase (TdT) deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) is the method of using the TdT enzyme to covalently ...

In Vivo Alkaline Comet Assay and Enzyme-modified Alkaline Comet Assay for Measuring DNA Strand Breaks and Oxidative DNA Damage in Rat Liver

Unrepaired DNA damage can lead to genetic instability, which in turn may enhance cancer development. Therefore, identifying potential DNA damaging agents is important for protecting public health. The in vivo alkaline comet assay, which detects DNA damage as strand breaks, is especially relevant for assessing the genotoxic hazards of xenobiotics, as its responses reflect the in vivo absorption, tissue distribution, metabolism and excretion (ADME) of chemicals, as well as DNA repair process. Compared to other in vivo DNA damage assays, the assay is rapid, sensitive, visual and inexpensive, and, by converting oxidative DNA damage into strand breaks using specific repair enzymes, the assay can measure oxidative DNA damage in an efficient and relatively artifact-free manner. Measurement of DNA damage with the comet assay can be performed using both acute and subchronic toxicology study designs, and by integrating the comet assay with other toxicological assessments, the assay addresses animal welfare requirements by making maximum use of animal resources. Another major advantage of the assays is that they only require a small amount of cells, and the cells do not have to be derived from proliferating cell populations. The assays also can be performed with a variety of human samples obtained from clinically or occupationally exposed individuals.

In Vivo Alkaline Comet Assay and Enzyme-modified Alkaline Comet Assay for Measuring DNA Strand Breaks and Oxidative DNA Damage in Rat Liver

The alkaline comet assay measures DNA strand breaks in eukaryotic cells. By adding an Endonuclease III or human 8-oxoguanine-DNA-N-glycosylase digestion step, the assay can efficiently detect oxidative DNA damage. We describe methods for using these assays to detect DNA damage in rat liver.

体内碱性彗星试验和酶修饰碱性彗星试验测量DNA链断裂和DNA氧化损伤大鼠肝

Unrepaired DNA damage can lead to genetic instability, which in turn may enhance cancer development. Therefore, identifying potential DNA damaging agents ...

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a versatile analytical tool for characterizing viscoelastic mechanical properties for a variety of materials ranging from hard (plastic, glass, metal, etc.) surfaces to cells on any substrate. It has been widely used to measure the stiffness of cells, but less frequently used to measure the stiffness of aortas. In this paper, we will describe the procedures for using AFM in contact mode to measure the ex vivo elastic modulus of unloaded mouse arteries. We describe our procedure for isolation of mouse aortas, and then provide detailed information for the AFM analysis. This includes step-by-step instructions for alignment of the laser beam, calibration of the spring constant and deflection sensitivity of the AFM probe, and acquisition of force curves. We also provide a detailed protocol for data analysis of the force curves.

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

We present detailed protocols for isolation of aortas from mouse and measurement of their elastic modulus using atomic force microscopy.

测量的刚度<em&gt;离体</em&gt;鼠标主动脉用原子力显微镜

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a ...

Demonstration of the DNA Fiber Assay for Investigating DNA Damage and Repair Dynamics Induced by Nanoparticles

Nanomaterial exposure can cause replication stress and genomic instability in cells. The degree of instability depends on the chemistry, size, and concentration of the nanomaterials, the time of exposure, and the exposed cell type. Several established methods have been used to elucidate how endogenous/exogenous agents impact global replication. However, replicon-level assays, such as the DNA fiber assay, are imperative to understand how these agents influence replication initiation, terminations, and replication fork progression. Knowing this allows one to understand better how nanomaterials increase the chances of mutation fixation and genomic instability. We used RAW 264.7 macrophages as model cells to study the replication dynamics under graphene oxide nanoparticle exposure. Here, we demonstrate the basic protocol for the DNA fiber assay, which includes pulse labeling with nucleotide analogs, cell lysis, spreading the pulse-labeled DNA fibers onto slides, fluorescent immunostaining of the nucleotide analogs within the DNA fibers, imaging of the replication intermediates within the DNA fibers using confocal microscopy, and replication intermediate analysis utilizing a computer-assisted scoring and analysis (CASA) software.

The methodology assists in the analysis of variation in DNA replication dynamics in the presence of nanoparticles. Different methodologies can be adopted based on the cytotoxicity level of the material of interest. In addition, a description of the image analysis is provided to help in DNA fiber analysis.