Name: measuring dna damage and repair in mouse splenocytes after chronic in vivo exposure to very low doses of beta- and gamma-radiation
Uploaded: 2015-07-03T17:00:00.000+00:00
Duration: 11 min 24 s
Description: 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

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>

<ol>
	<li>Mitchel, R. E., Jackson, J. S., McCann, R. A., Boreham, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+adaptive+response+modifies+latency+for+radiation-induced+myeloid+leukemia+in+CBA/H+mice.">The adaptive response modifies latency for radiation-induced myeloid leukemia in CBA/H mice.</a> Radiat Res. 152 (3), 273-279 (1999).</li><li>Shadley, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosomal+adaptive+response+in+human+lymphocytes.">Chromosomal adaptive response in human lymphocytes.</a> Radiat Res. 138 (Suppl 1), S9-12 (1994).</li><li>Wiencke, J. K., Afzal, V., Olivieri, G., Wolff, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+that+the+[3H]thymidine-induced+adaptive+response+of+human+lymphocytes+to+subsequent+doses+of+X-rays+involves+the+induction+of+a+chromosomal+repair+mechanism).">Evidence that the [3H]thymidine-induced adaptive response of human lymphocytes to subsequent doses of X-rays involves the induction of a chromosomal repair mechanism).</a> Mutagenesis. 1 (5), 375-380 (1986).</li><li>Mitchel, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dose+window+for+radiation-induced+protective+adaptive+responses.">The dose window for radiation-induced protective adaptive responses.</a> Dose-Response. 8 (2), 192-208 (2010).</li><li>Tubiana, M., Feinendegen, L. E., Yang, C., Kaminski, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+linear+no-threshold+relationship+is+inconsistent+with+radiation+biologic+and+experimental+data.">The linear no-threshold relationship is inconsistent with radiation biologic and experimental data.</a> Radiology. 251 (1), 13-22 (2009).</li><li>Gent, D. C., Hoeijmakers, J. H., Kanaar, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosomal+stability+and+the+DNA+double-stranded+break+connection.">Chromosomal stability and the DNA double-stranded break connection.</a> Nat Rev Genet. 2 (3), 196-206 (2001).</li><li>Jackson, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensing+and+repairing+DNA+double-strand+breaks.">Sensing and repairing DNA double-strand breaks.</a> Carcinogenesis. 23 (5), 687-696 (2002).</li><li>Rogakou, E. P., Pilch, D. R., Orr, A. H., Ivanova, V. S., Bonner, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+double-stranded+breaks+induce+histone+H2AX+phosphorylation+on+serine+139.">DNA double-stranded breaks induce histone H2AX phosphorylation on serine 139.</a> J Biol Chem. 273 (10), 5858-5868 (1998).</li><li>Schultz, L. B., Chehab, N. H., Malikzay, A., Halazonetis, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=p53+binding+protein+1+(53BP1)+is+an+early+participant+in+the+cellular+response+to+DNA+double-strand+breaks.">p53 binding protein 1 (53BP1) is an early participant in the cellular response to DNA double-strand breaks.</a> J Cell Biol. 151 (7), 1381-1390 (2000).</li><li>Kinner, A., Wu, W., Staudt, C., Iliakis, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gamma-H2AX+in+recognition+and+signaling+of+DNA+double-strand+breaks+in+the+context+of+chromatin.">Gamma-H2AX in recognition and signaling of DNA double-strand breaks in the context of chromatin.</a> Nucleic Acids Res. 36 (17), 5678-5694 (2008).</li><li>Sedelnikova, O. A., Rogakou, E. P., Panyutin, I. G., Bonner, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+detection+of+(125)IdU-induced+DNA+double-strand+breaks+with+gamma-H2AX+antibody.">Quantitative detection of (125)IdU-induced DNA double-strand breaks with gamma-H2AX antibody.</a> Radiat Res. 158 (4), 486-492 (2002).</li><li>Yuan, J., Adamski, R., Chen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Focus+on+histone+variant+H2AX:+to+be+or+not+to+be.">Focus on histone variant H2AX: to be or not to be.</a> FEBS Lett. 584 (17), 3717-3724 (2010).</li><li>Taneja, 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=Histone+H2AX+phosphorylation+as+a+predictor+of+radiosensitivity+and+target+for+radiotherapy.">Histone H2AX phosphorylation as a predictor of radiosensitivity and target for radiotherapy.</a> J Biol Chem. 279 (3), 2273-2280 (2004).</li><li>Olive, P. L., Banath, J. P., Sinnott, L. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphorylated+histone+H2AX+in+spheroids,+tumors,+and+tissues+of+mice+exposed+to+etoposide+and+3-amino-1,2,4-benzotriazine-1,3-dioxide.">Phosphorylated histone H2AX in spheroids, tumors, and tissues of mice exposed to etoposide and 3-amino-1,2,4-benzotriazine-1,3-dioxide.</a> Cancer Res. 64 (15), 5363-5369 (2004).</li><li>Banath, J. P., Klokov, D., MacPhail, S. H., Banuelos, C. A., Olive, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Residual+gammaH2AX+foci+as+an+indication+of+lethal+DNA+lesions.">Residual gammaH2AX foci as an indication of lethal DNA lesions.</a> BMC Cancer. 10, 4 (2010).</li><li>Blimkie, M. S., Fung, L. C., Petoukhov, E. S., Girard, C., Klokov, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repair+of+DNA+double-strand+breaks+is+not+modulated+by+low-dose+gamma+radiation+in+C57BL/6J+mice.">Repair of DNA double-strand breaks is not modulated by low-dose gamma radiation in C57BL/6J mice.</a> Radiat Res. 1815 (5), 548-559 (2014).</li><li>Runge, R., 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=Fully+automated+interpretation+of+ionizing+radiation-induced+gammaH2AX+foci+by+the+novel+pattern+recognition+system+AKLIDES(R).">Fully automated interpretation of ionizing radiation-induced gammaH2AX foci by the novel pattern recognition system AKLIDES(R).</a> Int J Radiat Biol. 88 (5), 439-447 (2012).</li><li>Jucha, A., 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=FociCounter:+A+freely+available+PC+programme+for+quantitative+and+qualitative+analysis+of+gamma-H2AX+foci.">FociCounter: A freely available PC programme for quantitative and qualitative analysis of gamma-H2AX foci.</a> Mutat Res. 696 (1), 16-20 (2010).</li><li>Garty, G., 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=The+RABIT:+a+rapid+automated+biodosimetry+tool+for+radiological+triage.">The RABIT: a rapid automated biodosimetry tool for radiological triage.</a> Health Phys. 98 (2), 209-217 (2010).</li><li>Kovalchuk, I. P., 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=Age-dependent+changes+in+DNA+repair+in+radiation-exposed+mice.">Age-dependent changes in DNA repair in radiation-exposed mice.</a> Radiat Res. 182 (6), 683-694 (2014).</li><li>Rube, C. E., 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=DNA+repair+in+the+context+of+chromatin:+new+molecular+insights+by+the+nanoscale+detection+of+DNA+repair+complexes+using+transmission+electron+microscopy.">DNA repair in the context of chromatin: new molecular insights by the nanoscale detection of DNA repair complexes using transmission electron microscopy.</a> DNA Repair. 10 (4), 427-437 (2011).</li><li>Amundson, S. A., Do, K. T., Fornace, A. 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><tbody><tr><td>HTO: tritiated water, [3H]</td><td>locally obtained from a nuclear reactor&amp;nbsp;</td><td></td><td>stock activity 3.7 GBq/mL;&amp;nbsp; 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>1 mCi/ml (185 MBq)</td></tr><tr><td>OBT: Glycine, [2-3H]: organically bound tritium</td><td>Perkin Elmer</td><td>NET004005MC</td><td>1 mCi/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>1 mCi/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&amp;nbsp;&amp;nbsp;</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&amp;nbsp;</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>1 mg/ml</td></tr><tr><td>ethanol</td><td>Commercial Alcohols</td><td>P006-EAAN</td><td>500 ml 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 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 12 cm sharp/sharp</td></tr><tr><td>forceps, straight</td><td>Fine Science Tools</td><td>11008-13</td><td>Semken forceps 13 cm, 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 60 x 15 mm</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>PBS recipe: 1 tablet dissolved in 200 ml 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</td><td></td><td></td><td></td></tr><tr><td>30 g TRIS HCl</td><td>Sigma Aldrich</td><td>T3253-1KG</td><td>Trizma hydrochloride</td></tr><tr><td>88 g NaCl</td><td>Fisher Scientific</td><td>S271-500</td><td>Sodium Chloride</td></tr><tr><td>2 g KCl</td><td>Fisher Scientific</td><td>P217-500</td><td>Potassium Chloride</td></tr><tr><td>Dissolve in 1 L 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

Universidad de Cartagena UDEC

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

Application of Laser Micro-irradiation for Examination of Single and Double Strand Break Repair in Mammalian Cells

Highly coordinated DNA repair pathways exist to detect, excise and replace damaged DNA bases, and coordinate repair of DNA strand breaks. While molecular biology techniques have clarified structure, enzymatic functions, and kinetics of repair proteins, there is still a need to understand how repair is coordinated within the nucleus. Laser micro-irradiation offers a powerful tool for inducing DNA damage and monitoring the recruitment of repair proteins. Induction of DNA damage by laser micro-irradiation can occur with a range of wavelengths, and users can reliably induce single strand breaks, base lesions and double strand breaks with a range of doses. Here, laser micro-irradiation is used to examine repair of single and double strand breaks induced by two common confocal laser wavelengths, 355 nm and 405 nm. Further, proper characterization of the applied laser dose for inducing specific damage mixtures is described, so users can reproducibly perform laser micro-irradiation data acquisition and analysis.

Confocal fluorescence microscopy and laser micro-irradiation offer tools for inducing DNA damage and monitoring the response of DNA repair proteins in selected sub-nuclear areas. This technique has significantly advanced our knowledge of damage detection, signaling, and recruitment. This manuscript demonstrates these technologies to examine single and double strand break repair.

激光微辐照在哺乳动物细胞单、双链断裂修复中的应用

Highly coordinated DNA repair pathways exist to detect, excise and replace damaged DNA bases, and coordinate repair of DNA strand breaks. While molecular ...

科研

癌症研究

Laser Microirradiation to Study In Vivo Cellular Responses to Simple and Complex DNA Damage

DNA damage induces specific signaling and repair responses in the cell, which is critical for protection of genome integrity. Laser microirradiation became a valuable experimental tool to investigate the DNA damage response (DDR) in vivo. It allows real-time high-resolution single-cell analysis of macromolecular dynamics in response to laser-induced damage confined to a submicrometer region in the cell nucleus. However, various laser conditions have been used without appreciation of differences in the types of damage induced. As a result, the nature of the damage is often not well characterized or controlled, causing apparent inconsistencies in the recruitment or modification profiles. We demonstrated that different irradiation conditions (i.e., different wavelengths as well as different input powers (irradiances) of a femtosecond (fs) near-infrared (NIR) laser) induced distinct DDR and repair protein assemblies. This reflects the type of DNA damage produced. This protocol describes how titration of laser input power allows induction of different amounts and complexities of DNA damage, which can easily be monitored by detection of base and crosslinking damages, differential poly (ADP-ribose) (PAR) signaling, and pathway-specific repair factor assemblies at damage sites. Once the damage conditions are determined, it is possible to investigate the effects of different damage complexity and differential damage signaling as well as depletion of upstream factor(s) on any factor of interest.

Laser Microirradiation to Study In Vivo Cellular Responses to Simple and Complex DNA Damage

The goal of this protocol is to describe how to use laser microirradiation to induce different types of DNA damage, including relatively simple strand breaks and complex damage, to study DNA damage signaling and repair factor assembly at damage sites in vivo.

激光 Microirradiation 研究体内细胞对简单复杂 DNA 损伤的反应

DNA damage induces specific signaling and repair responses in the cell, which is critical for protection of genome integrity. Laser microirradiation ...

遗传学

Immunofluorescence Microscopy of &#947;H2AX and 53BP1 for Analyzing the Formation and Repair of DNA Double-strand Breaks

DNA double-strand breaks (DSB) are serious DNA lesions. Analysis of the formation and repair of DSB is relevant in a broad spectrum of research areas including genome integrity, genotoxicity, radiation biology, aging, cancer, and drug development. In response to DSB, the histone H2AX is phosphorylated at Serine 139 in a region of several megabase pairs forming discrete nuclear foci detectable by immunofluorescence microscopy. In addition, 53BP1 (p53 binding protein 1) is another important DSB-responsive protein promoting repair of DSB by nonhomologous end-joining while preventing homologous recombination. According to the specific functions of &#947;H2AX and 53BP1, the combined analysis of &#947;H2AX and 53BP1 by immunofluorescence microscopy may be a reasonable approach for a detailed analysis of DSB. This manuscript provides a step-by-step protocol supplemented with methodical notes for performing the technique. Specifically, the influence of the cell cycle on &#947;H2AX foci patterns is demonstrated in normal fibroblasts of the cell line NHDF. Further, the value of the &#947;H2AX foci as a biomarker is depicted in x-ray irradiated lymphocytes of a healthy individual. Finally, genetic instability is investigated in CD34+ cells of a patient with acute myeloid leukemia by immunofluorescence microscopy of &#947;H2AX and 53BP1.

This manuscript provides a protocol for the analysis of DNA double-strand breaks by immunofluorescence microscopy of &#947;H2AX and 53BP1.

γH2AX 和53BP1 的免疫荧光显微镜分析 DNA 双链断裂的形成和修复

DNA double-strand breaks (DSB) are serious DNA lesions. Analysis of the formation and repair of DSB is relevant in a broad spectrum of research areas ...

CometChip: A High-throughput 96-Well Platform for Measuring DNA Damage in Microarrayed Human Cells

DNA damaging agents can promote aging, disease and cancer and they are ubiquitous in the environment and produced within human cells as normal cellular metabolites. Ironically, at high doses DNA damaging agents are also used to treat cancer. The ability to quantify DNA damage responses is thus critical in the public health, pharmaceutical and clinical domains. Here, we describe a novel platform that exploits microfabrication techniques to pattern cells in a fixed microarray. The &#8216;CometChip&#8217; is based upon the well-established single cell gel electrophoresis assay (a.k.a. the comet assay), which estimates the level of DNA damage by evaluating the extent of DNA migration through a matrix in an electrical field. The type of damage measured by this assay includes abasic sites, crosslinks, and strand breaks. Instead of being randomly dispersed in agarose in the traditional assay, cells are captured into an agarose microwell array by gravity. The platform also expands from the size of a standard microscope slide to a 96-well format, enabling parallel processing. Here we describe the protocols of using the chip to evaluate DNA damage caused by known genotoxic agents and the cellular repair response followed after exposure. Through the integration of biological and engineering principles, this method potentiates robust and sensitive measurements of DNA damage in human cells and provides the necessary throughput for genotoxicity testing, drug development, epidemiological studies and clinical assays.

We describe here a platform that allows comet assay detection of DNA damage with unprecedented throughput. The device patterns mammalian cells into a microarray and enables parallel processing of 96 samples. The approach facilitates analysis of base level DNA damage, exposure-induced DNA damage and DNA repair kinetics.

CometChip：一种高通量的96孔平台的微阵列人类细胞测量DNA损伤

DNA damaging agents can promote aging, disease and cancer and they are ubiquitous in the environment and produced within human cells as normal cellular ...

生物工程

Identifying DNA Mutations in Purified Hematopoietic Stem/Progenitor Cells

In recent years, it has become apparent that genomic instability is tightly related to many developmental disorders, cancers, and aging. Given that stem cells are responsible for ensuring tissue homeostasis and repair throughout life, it is reasonable to hypothesize that the stem cell population is critical for preserving genomic integrity of tissues. Therefore, significant interest has arisen in assessing the impact of endogenous and environmental factors on genomic integrity in stem cells and their progeny, aiming to understand the etiology of stem-cell based diseases.
LacI transgenic mice carry a recoverable &#955;&#160;phage vector encoding the LacI reporter system, in which the LacI gene serves as the mutation reporter. The result of a mutated LacI gene is the production of &#946;-galactosidase that cleaves a chromogenic substrate, turning it blue. The LacI reporter system is carried in all cells, including stem/progenitor cells and can easily be recovered and used to subsequently infect E. coli. After incubating infected E. coli on agarose that contains the correct substrate, plaques can be scored; blue plaques indicate a mutant LacI gene, while clear plaques harbor wild-type. The frequency of blue (among clear) plaques indicates the mutant frequency in the original cell population the DNA was extracted from. Sequencing the mutant LacI gene will show the location of the mutations in the gene and the type of mutation.
The LacI transgenic mouse model is well-established as an in vivo mutagenesis assay. Moreover, the mice and the reagents for the assay are commercially available. Here we describe in detail how this model can be adapted to measure the frequency of spontaneously occurring DNA mutants in stem cell-enriched Lin-IL7R-Sca-1+cKit++(LSK) cells and other subpopulations of the hematopoietic system.

Here we describe an in vivo mutagenesis assay for small numbers of purified hematopoietic cells using the LacI transgenic mouse model.&#160;The LacI gene can be isolated to determine the frequency, location, and type of DNA mutants&#160;spontaneously arisen or after exposure to genotoxins.

在纯化造血干/祖细胞识别DNA突变

In recent years, it has become apparent that genomic instability is tightly related to many developmental disorders, cancers, and aging. Given that stem ...

免疫与感染

Dual Immunofluorescence of &#947;H2AX and 53BP1 in Human Peripheral Lymphocytes

Double strand breaks (DSBs) are one of the most severe lesions that can occur in cell nuclei, and, if not repaired, they can lead to severe outcomes, including cancer. The cell is, therefore, provided with complex mechanisms to repair DSBs, and these pathways involve histone H2AX in its phosphorylated form at Ser-139 (namely &#947;H2AX) and p53 binding protein 1 (53BP1). As both proteins can form foci at the sites of DSBs, identification of these markers is considered a suitable method to study both DSBs and their kinetics of repair. According to the molecular processes that lead to the formation of &#947;H2AX and 53BP1 foci, it could be more useful to investigate their co-localization near the DSBs in order to set up an alternative approach that allows quantifying DSBs by the simultaneous detection of two DNA damage markers. Thus, this protocol aims to assess the genomic damage induced in human lymphocytes by the radiomimetic agent bleomycin through the presence of &#947;H2AX and 53BP1 foci in a dual immunofluorescence. Using this methodology, we also delineated the variation in the number of &#947;H2AX and 53BP1 foci over time, as a preliminary attempt to study the repair kinetics of bleomycin-induced DSBs.

This protocol presents a method to assess the formation and repair of DNA double-strand breaks through the simultaneous detection of &#947;H2AX and 53BP1 foci in interphase nuclei of bleomycin-treated human peripheral lymphocytes.

人外周淋巴细胞中γH2AX和53BP1的双重免疫荧光

Double strand breaks (DSBs) are one of the most severe lesions that can occur in cell nuclei, and, if not repaired, they can lead to severe outcomes, ...

Assessing Cell Cycle Progression of Neural Stem and Progenitor Cells in the Mouse Developing Brain after Genotoxic Stress

Neurons of the cerebral cortex are generated during brain development from different types of neural stem and progenitor cells (NSPC), which form a pseudostratified epithelium lining the lateral ventricles of the embryonic brain. Genotoxic stresses, such as ionizing radiation, have highly deleterious effects on the developing brain related to the high sensitivity of NSPC. Elucidation of the cellular and molecular mechanisms involved depends on the characterization of the DNA damage response of these particular types of cells, which requires an accurate method to determine NSPC progression through the cell cycle in the damaged tissue. Here is shown a method based on successive intraperitoneal injections of EdU and BrdU in pregnant mice and further detection of these two thymidine analogues in coronal sections of the embryonic brain. EdU and BrdU are both incorporated in DNA of replicating cells during S phase and are detected by two different techniques (azide or a specific antibody, respectively), which facilitate their simultaneous detection. EdU and BrdU staining are then determined for each NSPC nucleus in function of its distance from the ventricular margin in a standard region of the dorsal telencephalon. Thus this dual labeling technique allows distinguishing cells that progressed through the cell cycle from those that have activated a cell cycle checkpoint leading to cell cycle arrest in response to DNA damage.
An example of experiment is presented, in which EdU was injected before irradiation and BrdU immediately after and analyzes performed within the 4 hr&#160;following irradiation. This protocol provides an accurate analysis of the acute DNA damage response of NSPC in function of the phase of the cell cycle at which they have been irradiated. This method is easily transposable to many other systems in order to determine the impact of a particular treatment on cell cycle progression in living tissues.

Administration of two analogs of thymidine, EdU and BrdU, in pregnant mice allows the analysis of cell cycle progression in neural and progenitor cells in the embryonic mouse brain. This method is useful to determine the effects of genotoxic stress, including ionizing radiation, during brain development.

神经干细胞和祖细胞在小鼠脑开发基因毒性应激后的评估细胞周期进程

Neurons of the cerebral cortex are generated during brain development from different types of neural stem and progenitor cells (NSPC), which form a ...

神经科学

Quantitation of &gamma;H2AX Foci in Tissue Samples

DNA double-strand breaks (DSBs) are particularly lethal and genotoxic lesions, that can arise either by endogenous (physiological or pathological) processes or by exogenous factors, particularly ionizing radiation and radiomimetic compounds. Phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX, is an early response to DNA double-strand breaks1. This phosphorylation event is mediated by the phosphatidyl-inosito 3-kinase (PI3K) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Overall, DSB induction results in the formation of discrete nuclear &gamma;H2AX foci which can be easily detected and quantitated by immunofluorescence microscopy2. Given the unique specificity and sensitivity of this marker, analysis of &gamma;H2AX foci has led to a wide range of applications in biomedical research, particularly in radiation biology and nuclear medicine. The quantitation of &gamma;H2AX foci has been most widely investigated in cell culture systems in the context of ionizing radiation-induced DSBs. Apart from cellular radiosensitivity, immunofluorescence based assays have also been used to evaluate the efficacy of radiation-modifying compounds. In addition, &gamma;H2AX has been used as a molecular marker to examine the efficacy of various DSB-inducing compounds and is recently being heralded as important marker of ageing and disease, particularly cancer3. Further, immunofluorescence-based methods have been adapted to suit detection and quantitation of &gamma;H2AX foci ex vivo and in vivo4,5. Here, we demonstrate a typical immunofluorescence method for detection and quantitation of &gamma;H2AX foci in mouse tissues.

Quantitation of &#947;H2AX Foci in Tissue Samples

Quantitation of DNA double-strand breaks on the basis of &gamma;H2AX foci has become an invaluable tool, particularly in radiation biology, for the evaluation of tissue radiosensitivity and effects of radiation modifying compounds. Here we demonstrate the use of an immunofluorescence assay for quantitation of &gamma;H2AX foci in tissue samples.

定量组织样本中的γH2AX灶

DNA double-strand breaks (DSBs) are particularly lethal and genotoxic lesions, that can arise either by endogenous (physiological or pathological) ...

生物学

Diffuse Optical Spectroscopy for the Quantitative Assessment of Acute Ionizing Radiation Induced Skin Toxicity Using a Mouse Model

Acute skin toxicities from ionizing radiation (IR) are a common side effect from therapeutic courses of external beam radiation therapy (RT) and negatively impact patient quality of life and long term survival. Advances in the understanding of the biological pathways associated with normal tissue toxicities have allowed for the development of interventional drugs, however, current response studies are limited by a lack of quantitative metrics for assessing the severity of skin reactions. Here we present a diffuse optical spectroscopic (DOS) approach that provides quantitative optical biomarkers of skin response to radiation. We describe the instrumentation design of the DOS system as well as the inversion algorithm for extracting the optical parameters. Finally, to demonstrate clinical utility, we present representative data from a pre-clinical mouse model of radiation induced erythema and compare the results with a commonly employed visual scoring. The described DOS method offers an objective, high through-put evaluation of skin toxicity via functional response that is translatable to the clinical setting.

We present a diffuse optical spectroscopic (DOS) approach that provides quantitative optical biomarkers of skin response to radiation. We describe DOS instrumentation design, optical parameters extraction algorithms and the animal handling procedures required to yield representative data from a pre-clinical mouse model of radiation induced erythema.

弥漫性光学光谱治疗急性电离辐射引起的皮肤毒性的定量评价使用小鼠模型

Acute skin toxicities from ionizing radiation (IR) are a common side effect from therapeutic courses of external beam radiation therapy (RT) and ...

医学

Preparation of Peripheral Blood Mononuclear Cell Pellets and Plasma from a Single Blood Draw at Clinical Trial Sites for Biomarker Analysis

Analysis of biomarkers in peripheral blood is becoming increasingly important in clinical trials to establish proof of mechanism to evaluate effects of treatment, and help guide dose and schedule setting of therapeutics. From a single blood draw, peripheral blood mononuclear cells can be isolated and processed to analyze and quantify protein markers, and plasma samples can be used for the analysis of circulating tumor DNA, cytokines, and plasma metabolomics. Longitudinal samples from a treatment provide information on the evolution of a given protein marker, the mutational status and immunological landscape of the patient. This can only be achieved if the processing of the peripheral blood is carried out effectively in clinical sites and samples are properly preserved from the bedside to bench. Here, we present an optimized general-purpose protocol that can be implemented at clinical sites for obtaining PBMC pellets and plasma samples in multi-center clinical trials, that will enable clinical professionals in hospital laboratories to successfully provide high quality samples, regardless of their level of technical expertise. Alternative protocol variations are also presented that are optimized for more specific downstream analytical methods. We apply this protocol for studying protein biomarkers against DNA damage response (DDR) on X-ray irradiated blood to demonstrate the suitability of the approach in oncology settings where DDR drugs and/or radiotherapy have been practiced as well as in preclinical stages where mechanistic hypothesis testing is required.

This protocol details clinically implementable preparation of high quality PBMC and plasma biosamples at the clinical trial site that can be used for translational biomarker analysis.

在生物标志物分析临床试验场单次抽血时，准备外周血单核细胞颗粒和血浆

Analysis of biomarkers in peripheral blood is becoming increasingly important in clinical trials to establish proof of mechanism to evaluate effects of ...

测量DNA损伤与修复小鼠脾细胞慢性后

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