作者感谢 L. Pieters、T. Thiron 和 G. De Smet 的技术支持。我们感谢所有为这项研究献血的志愿者。这项工作得到了格兰特（T000118N）下佛兰德斯研究基金会（FWO）的财政支持。

在这项研究中，通过静脉穿刺收集了 30 名年龄在 17 至 65 岁之间的健康供体的血样。来自 20 名捐献者的 MN 数据从 Beyls 等人的论文中重复使用8 血液 样本的收集符合比利时根特大学医院伦理委员会（注册号:2019/1565）的指南。已获得所有参与者的书面知情同意书。有关本协议中使用的所有材料和试剂的详细信息 ，请参阅材料表 。
1. 采集血样
<ol><li>在开始之前，确保参与者的书面知情同意书可用，并正确遵守道德准则。</li>
	<li>将血液收集到Li-肝素管中并在室温下储存，直到准备好进行冷冻保存。</li>
</ol>2. 全血的冷冻保存
注意:细胞收获之前的所有步骤都在无菌层流气流中无菌进行。
<ol><li>要冷冻保存 1 mL 血液，通过混合 800 μL 胎牛血清 （FCS） 和 200 μL 二甲基亚砜 （DMSO） 制备 1 mL 冷冻培养基。 注意:冷冻培养基的制备体积等于需要冷冻的血液样本。</li>
	<li>对于全血的冷冻保存，将血液样本从肝素管转移到 15 或 50 mL 离心管中 注意:管子的容量取决于血液的体积。</li>
	<li>以低速涡旋血液，连续但 滴加 等体积的冷冻培养基。</li>
	<li>将 2 mL 血液冷冻混合物转移到冷冻管 （2 mL） 中，其中每个等分试样有 1 mL 血液样本与 1 mL 冷冻混合物混合。</li>
	<li>逐步逐步冻结<ol><li>将冷冻管转移到含有异丙醇的冷冻箱中，并放入冰箱（-80°C）过夜。</li>
		<li>将冷冻管转移到液氮中长期使用。</li>
	</ol></li>
</ol>3. 冷冻保存的血液解冻
注意:为了限制解冻过程的总时间，一次最多应处理 8 个冻存管（每个 2 mL）。
<ol><li>制备<ol><li>在解冻血液之前，将200 mL无菌磷酸盐缓冲盐水（PBS）预热至37°C。</li>
		<li>在无菌条件下制备所需培养基的工作溶液。确保每次培养的培养基含有 1.6 mL 完全培养基:补充有青霉素/链霉素 （0.5%）、0.2 mL FCS、20 μL 丙酮酸钠和 2 μL β-巯基乙醇的罗斯韦尔公园纪念研究所 （RPMI） 培养基。 注意:由于解冻时可能会丢失大量血细胞，因此解冻的血液在总体积为 2 mL 的 24 孔板等小孔中培养。每剂使用一个多孔板。</li>
		<li>对于每个培养孔，在盖子上注明供体代码和A / B以及日期和剂量，如下所示（图1）。</li>
	</ol></li>
</ol><img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65855/65855fig01.jpg"> 图 1:用于培养用于微核测定的全血的 24 孔板的建议布局。 标记孔并在盖子上的适当位置标明供体代码。对于每个患者样本，重复的孔应彼此相邻。请注意，它是 10 x 10 准直器的建议布局，可以根据准直器或样品的大小进行更改。 <a href="https://www.jove.com/files/ftp_upload/65855/65855fig01large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<ol start="2"><li>解冻和培养全血的步骤<ol><li>将水浴加热至37°C。</li>
		<li>从液氮中取出所需的冷冻管（数量取决于剂量等要求）。 注意:一个冷冻管含有 1 mL 血液和 1 mL 冷冻培养基。因此，可以用来自一个冷冻卵管的血液建立两个培养物。</li>
		<li>在温水浴中部分解冻冷冻管（直到仍然看到小冰晶）。</li>
		<li>从水浴中取出小瓶，并用无菌纸巾清洁小瓶的外部。</li>
		<li>在无菌层流罩中，非常缓慢地打开小瓶，以防止管内形成的气泡溢出。</li>
		<li>重悬每个小瓶的内容物，并将它们单独转移到 15 mL 锥形离心管（每小瓶 1 管）中。</li>
		<li>向相应的试管中滴加 1 mL 温热 （37 °C） PBS，同时轻轻旋转试管。用 P1000 移液器重悬。</li>
		<li>为了稀释和均匀洗涤，滴加7 mL预热的PBS（37°C），并用P1000移液管重悬。</li>
		<li>在室温下以180× g （设置为 加速1，制动1）离心血液8分钟。</li>
		<li>在此等待步骤中，用 500 μL PBS 填充空孔（将要培养细胞的孔除外）。</li>
		<li>将孔板放回培养箱中预热。</li>
		<li>离心完成后，小心地除去上清液，留下约1 mL，以避免干扰细胞沉淀。</li>
		<li>用P1000移液管将沉淀重悬于剩余的上清液中。</li>
		<li>将8mL温热的PBS（37°C）滴加到相应的试管中，同时轻轻旋转试管。然后，用P1000移液器重悬。</li>
		<li>将重悬的血液以180× g 离心8分钟（ 设置为加速度9，制动9）。</li>
		<li>在此等待步骤中，将 200 μL 培养基转移到每个标记的孔中。</li>
		<li>将板放回培养箱中以保持37°C。</li>
		<li>离心后，以连续运动除去上清液（以避免干扰松散的细胞沉淀），但留下约80μL的上清液。</li>
		<li>向沉淀中加入 280 μL FCS（室温）并重悬（总体积为 360 μL）。</li>
		<li>将 180 μL 细胞悬液转移到每个孔中并重悬（= 0.5 mL "血液"/培养物）。现在，每个孔中的总体积为 380 μL，形成 2 mm 的培养基层。</li>
		<li>在辐照前，将板在37°C的CO2 培养箱中孵育至少10分钟。</li>
	</ol></li>
</ol>4. G0 MN测定
<ol><li>从培养箱中取出培养板，并在室温下用所需剂量（例如 0.5、1 和 2 Gy X 射线（220 kV、13 mA、0.15 mm Cu）照射细胞培养物。使用假辐照样品作为对照来鉴定自发的 MN 产量。</li>
	<li>辐照后，立即向每个孔中加入1.62mL培养基（37°C）。</li>
	<li>为了刺激 T 淋巴细胞中的细胞分裂，向每个孔中加入 40 μL 植物血凝素 （PHA） 并彻底重悬。</li>
	<li>将这些板放回培养箱（5%CO 2,37°C）。 注意:这将是测定的 开始时间 。</li>
	<li>通过每孔加入 8 μL 细胞松弛素 B （6 μg/mL） 在刺激后 23 小时阻断胞质分裂并适当重悬。</li>
	<li>在刺激/培养时间后70小时收获细胞培养物。将细胞重悬并转移到 15 mL 试管中。用 2 mL PBS 冲洗每个孔，并将该 PBS 加入相应的 15 mL 试管中。</li>
	<li>在室温下以180× g离心管8分钟。</li>
	<li>弃去上清液，但在沉淀上留下约500μL。</li>
	<li>涡旋沉淀（全速），在涡旋的同时，缓慢滴加2mL冷氯化钾（KCl，4°C）。</li>
	<li>立即将细胞以180× g 离心8分钟。</li>
	<li>弃去上清液，留下约500μL上清液。</li>
	<li>涡旋沉淀（全速），在涡旋的同时，缓慢滴加 2 mL 冷 固定剂 1 （甲醇/乙酸/林格溶液，比例为 4:1:5）。</li>
	<li>将样品管在4°C下过夜（最短12小时，最多96小时）。</li>
	<li>以180× g离心8分钟。</li>
	<li>弃去上清液。</li>
	<li>涡旋沉淀（全速），在涡旋的同时，缓慢滴加2mL冷 固定剂2 （甲醇/乙酸，比例为4:1）。</li>
	<li>以180× g离心8分钟。</li>
	<li>弃去上清液。</li>
	<li>涡旋沉淀（全速），然后在涡旋的同时缓慢（滴加）2 mL 冷 固定剂 2 到沉淀中。</li>
	<li>将样品管保持在4°C（最短12小时和最多96小时）。</li>
	<li>用异丙醇清洁载玻片并正确标记它们。 注意: 使用打印的贴纸贴标签，因为记号笔墨水在加工时很容易擦掉。</li>
	<li>将样品管以180× g离心8分钟。</li>
	<li>将上清液转移到另一个管中，以根据沉淀的大小浓缩细胞。</li>
	<li>涡旋颗粒。</li>
	<li>将 40 μL 固定细胞滴入干燥干净的载玻片上。</li>
	<li>让载玻片在室温下干燥。立即将载玻片存放在盒子或染色剂中以供观察。</li>
</ol>5.吖啶橙（AO）染色
<ol><li>将载玻片浸入AO染色剂中1分钟，然后在蒸馏水中快速洗涤，然后将载玻片置于磷酸盐缓冲液（pH 6.8）中1分钟。</li>
	<li>从缓冲溶液中取出载玻片，并用干净的薄纸，擦干载玻片的背面，然后将载玻片放在干净的薄纸上。</li>
	<li>将 20 μL 磷酸盐缓冲液滴在载玻片顶部，并用干净的盖玻片轻轻盖住，避免气泡。</li>
	<li>用硅胶粘合剂密封这些载玻片。 注意:由于 AO 对光敏感并且会随着时间的推移而褪色，因此为了获得良好的对比度和荧光，建议在染色后 5 天内对载玻片进行评分。未检查时，始终将载玻片存放在冷藏室（4°C）中。</li>
</ol>6. 对染色载玻片进行评分
<ol><li>将载玻片置于荧光显微镜下，检查 1,000 个双核细胞 （BN）。手动计算 MN，MN 是毒性的六种生物标志物之一。为此，请让两名独立的评分员在相同的放大倍率下检查 500 个 BN 细胞/载玻片。 注:以下是 Fenech 推荐的评分标准的一些亮点，用于对染色载玻片9 进行评分<ol><li>通过寻找具有完整细胞质的圆形细胞来选择 BN 细胞，这些细胞具有两个大小大致相同、形状规则和染色模式的明显区分的细胞核。确保BN细胞的细胞质与相邻细胞的细胞质可区分。</li>
		<li>对 MN 进行评分，注意它在形态上与主核相同但小于主核;即使是最大的MN也不能超过主核直径的1/3。只有当MN不接触主核或至少微核边界与主核边界可区分时，才对MN进行评分。</li>
	</ol></li>
</ol>

检查辐照全血样品中的微核产量

<ol>
	<li>Grade, M., Difilippantonio, M. J., Camps, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterns+of+chromosomal+aberrations+in+solid+tumors.">Patterns of chromosomal aberrations in solid tumors.</a> Recent Results in Cancer Research. 200, 115-142 (2015).</li><li>Jiao, Y., Cao, F., Liu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radiation-induced+cell+death+and+its+mechanisms.">Radiation-induced cell death and its mechanisms.</a> Health Physics. 123 (5), 376-386 (2022).</li><li>Roederer, M., 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+genetic+architecture+of+the+human+immune+system:+a+bioresource+for+autoimmunity+and+disease+pathogenesis.">The genetic architecture of the human immune system: a bioresource for autoimmunity and disease pathogenesis.</a> Cell. 161 (2), 387-403 (2015).</li><li>Bankoglu, E. 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=Effect+of+cryopreservation+on+DNA+damage+and+DNA+repair+activity+in+human+blood+samples+in+the+comet+assay.">Effect of cryopreservation on DNA damage and DNA repair activity in human blood samples in the comet assay.</a> Archives of Toxicology. 95, 1831-1841 (2021).</li><li>Zijno, A., Saini, F., Crebelli, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suitability+of+cryopreserved+isolated+lymphocytes+for+the+analysis+of+micronuclei+with+the+cytokinesis-block+method.">Suitability of cryopreserved isolated lymphocytes for the analysis of micronuclei with the cytokinesis-block method.</a> Mutagenesis. 22, 311-315 (2007).</li><li>Sioen, S., Cloet, K., Vral, A., Baeyens, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cytokinesis-block+micronucleus+assay+on+human+isolated+fresh+and+cryopreserved+peripheral+blood+mononuclear+cells.">The cytokinesis-block micronucleus assay on human isolated fresh and cryopreserved peripheral blood mononuclear cells.</a> Journal of Personalized Medicine. 10 (3), 125 (2020).</li><li>Cheng, L., Wang, L. E., Spitz, M. R., Wei, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryopreserving+whole+blood+for+functional+assays+using+viable+lymphocytes+in+molecular+epidemiology+studies.">Cryopreserving whole blood for functional assays using viable lymphocytes in molecular epidemiology studies.</a> Cancer Letters. 166 (2), 155-163 (2001).</li><li>Beyls, E., Baeyens, A., Vral, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cytokinesis-block+micronucleus+assay+for+cryopreserved+whole+blood.">The cytokinesis-block micronucleus assay for cryopreserved whole blood.</a> International Journal of Radiation Biology. 97 (9), 1252-1260 (2021).</li><li>Fenech, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytokinesis-block+micronucleus+cytome+assay.">Cytokinesis-block micronucleus cytome assay.</a> Nature Protocols. 2 (5), 1084-1104 (2007).</li><li>Ramachandran, H., 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=Optimal+thawing+of+cryopreserved+peripheral+blood+mononuclear+cells+for+use+in+high-throughput+human+immune+monitoring+studies.">Optimal thawing of cryopreserved peripheral blood mononuclear cells for use in high-throughput human immune monitoring studies.</a> Cells. 1 (3), 313-324 (2012).</li><li>H&#248;nge, B. L., Petersen, M. S., Olesen, R., M&#248;ller, B. K., Erikstrup, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimizing+recovery+of+frozen+human+peripheral+blood+mononuclear+cells+for+flow+cytometry.">Optimizing recovery of frozen human peripheral blood mononuclear cells for flow cytometry.</a> PLoS One. 12 (11), e0187440-e0187517 (2017).</li><li>Alam, I., Goldeck, D., Larbi, A., Pawelec, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometric+lymphocyte+subset+analysis+using+material+from+frozen+whole+blood.">Flow cytometric lymphocyte subset analysis using material from frozen whole blood.</a> Journal of Immunoassay and Immunochemistry. 33 (2), 128-139 (2012).</li><li>Langenski&#246;ld, C., Mellgren, K., Abrahamsson, J., Bemark, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+blood+cell+subtype+concentrations+from+frozen+whole+blood+samples+using+TruCount+beads.">Determination of blood cell subtype concentrations from frozen whole blood samples using TruCount beads.</a> Cytometry Part B: Clinical Cytometry. 94B, 660-666 (2018).</li><li>Braudeau, C., 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=An+easy+and+reliable+whole+blood+freezing+method+for+flow+cytometry+immuno-phenotyping+and+functional+analyses.">An easy and reliable whole blood freezing method for flow cytometry immuno-phenotyping and functional analyses.</a> Cytometry Part B: Clinical Cytometry. 100 (6), 652-665 (2021).</li><li>Thierens, H., Vral, A., De Ridder, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+dosimetry+using+the+micronucleus+assay+for+lymphocytes:+interindividual+differences+in+dose+response.">Biological dosimetry using the micronucleus assay for lymphocytes: interindividual differences in dose response.</a> Health Physics. 61 (5), 623-630 (1991).</li><li>Vral, A., Fenech, M., Thierens, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+micronucleus+assay+as+a+biological+dosimeter+of+in+vivo+ionising+radiation+exposure.">The micronucleus assay as a biological dosimeter of in vivo ionising radiation exposure.</a> Mutagenesis. 26 (1), 11-17 (2011).</li><li>Vral, A., Thierens, H., Baeyens, A., De Ridder, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+micronucleus+and+G2-phase+assays+for+human+blood+lymphocytes+as+biomarkers+of+individual+sensitivity+to+ionizing+radiation:+limitations+imposed+by+intraindividual+variability.">The micronucleus and G2-phase assays for human blood lymphocytes as biomarkers of individual sensitivity to ionizing radiation: limitations imposed by intraindividual variability.</a> Radiation Research. 157 (4), 472-477 (2002).</li><li>Pajic, J., 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=Inter-individual+variability+in+the+response+of+human+peripheral+blood+lymphocytes+to+ionizing+radiation:+comparison+of+the+dicentric+and+micronucleus+assays.">Inter-individual variability in the response of human peripheral blood lymphocytes to ionizing radiation: comparison of the dicentric and micronucleus assays.</a> Radiation and Environmental Biophysics. 54, 317-325 (2015).</li><li>Fenech, M., 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=Intra-+and+inter-laboratory+variation+in+the+scoring+of+micronuclei+and+nucleoplasmic+bridges+in+binucleated+human+lymphocytes:+results+of+an+international+slide-scoring+exercise+by+the+HUMN+project.">Intra- and inter-laboratory variation in the scoring of micronuclei and nucleoplasmic bridges in binucleated human lymphocytes: results of an international slide-scoring exercise by the HUMN project.</a> Mutation Research. 534 (1-2), 45-64 (2003).</li><li>Miszczyk, J., Rawoj&#263;, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+culturing+technique+on+human+peripheral+blood+lymphocytes+response+to+proton+and+X-ray+radiation.">Effects of culturing technique on human peripheral blood lymphocytes response to proton and X-ray radiation.</a> International Journal of Radiation Biology. 96 (4), 424-433 (2020).</li></ol>

作者没有利益冲突需要声明。

CBMN测定应用的改进方案是一种相对简单方便的散装储存血液样本的方法。该程序概述了在冷冻保存和 CBMN 检测过程中需要注意的所有微小但重要的细节。其他实验室方案通常在冷冻混合物中使用 10% DMSO，而我们的冷冻混合物含有 20% DMSO 和 80% FCS7。由于这种冷冻混合物以等体积添加到全血样品中，因此最终浓度也是 10% DMSO。为了提高细胞存活率并刺激细胞分裂，我们在完全培养基 （cRPMI） 中加入 1% 丙酮酸钠和 0.1% β-巯基乙醇。这符合我们研究小组 6,8 建立的细胞培养方案。
尽管在该方案中无法完全解决解冻时细胞聚集的问题，但细胞分离优于其他常规方案。在洗涤步骤中滴加预热的PBS（37°C）时，我们获得了更好的结果，而不是一致，突然添加培养基来恢复解冻后的细胞。这种改进有助于减少细胞应激，并最大限度地减少结块，并证明细胞回收率高。此外，当PBS与RPMI相比，在细胞活力方面没有观察到明显差异。该小组已经证实，冷冻保存期的长度（长达 1 年）不会影响辐照和非辐照样品的 MN 产率 6,8。研究表明，在低温（液氮）下逐渐冷冻，然后在解冻时逐渐添加预热培养基，可以实现良好的细胞活力和 PBMC 的增殖10,11。研究人员已经表明，解冻的全血中 T 淋巴细胞的亚群与解冻的 PBMC 中观察到的亚群相当12.此外，已证明从冷冻保存的全血样本中回收的细胞亚型与在新鲜全血样本中观察到的细胞亚型相似13,14。
如果我们检查报告中获得的指示性结果，我们会看到随剂量的线性二次增加（图3）与其他关于线性能量转移（LET）诱导的微核的文献报道一致15,16。在冷冻保存的全血样品中观察到的MN产量的变异性（表1）与新鲜全血培养物报告的变异性8,17,18相同。此处详细描述的方案已在 20 名健康志愿者的新鲜和冷冻保存的全血上进行了验证8.核分裂指数 （NDI） 是该报告8 中观察到的细胞增殖的重要参数，与建议的新鲜全血19,20 一致。综上所述，这种优化的全血冷冻保存方案和改良的微核测定提供了更好的细胞产量，因此，建议将该方案用于放射敏感性评估。由于该协议的可重复性已经得到验证8，因此建议在大规模和多中心研究中具有适用性。

自发现以来，电离辐射 （IR） 的使用一直是研究人员争论的问题，因为它对生物有不利影响。有害影响通常表现为 DNA 损伤，例如双链断裂 （DSB），并且无法修复这些 DSB 会导致染色体畸变和突变，这是癌症的重要标志 1,2。这种染色体畸变可以通过细胞源性测定来检查，例如胞质分裂阻滞微核 （CBMN） 测定。微核是滞后的染色体片段，不能合并到子核中，因此在有丝分裂过程中会留下。
CBMN 是一种常用的可靠细胞遗传学技术，用于评估暴露于体内或体外电离辐射的个体的染色体损伤。新鲜全血或分离的外周血单核细胞 （PBMC） 可用于 CBMN 检测。新鲜全血主要是首选的生物材料，因为PBMC的分离和处理可能非常耗时，并且伴随着作为细胞存活和生长支持介质的血清血浆的损失。为了获得良好的双核细胞产量，应在采集后立即处理新鲜全血。然而，在时间限制下，立即处理的需要可能在物流上具有挑战性。此外，当许多样本应该在很长一段时间内采集或在远离处理中心的地点采集时，新鲜血液样本的储存可能是一个限制因素3,4。
此外，为了允许在同一个体/患者中重复进行 MN 分析，冷冻血液样本将是有益的。储存淋巴细胞以供以后应用CBMN测定的一种方法是冷冻分离的PBMC 5,6。然而，这种技术在冻结PBMC之前需要几个处理步骤。因此，全血的冷冻保存将是分离的PBMC冷冻保存的一种简单且省时的替代方案。 关于使用冷冻全血进行细胞遗传学检测或需要淋巴细胞增殖的检测的信息很少。只有一篇论文报道了使用冷冻保存的全血进行中期分析7.
由于全血的冷冻保存在生物监测、生物剂量学和放射敏感性评估领域具有许多优势，因此我们小组优化了全血冷冻保存方案，允许应用CBMN 测定 8。我们证明，冷冻保存的全血培养物中存在的淋巴细胞保持其基因组完整性和增殖能力至少 1 年。在本文中，我们详细描述了由 Beyls 等人 8 优化的冷冻保存程序和 CBMN 测定方案，并报告了对 30 名健康个体的冷冻血液样本获得的结果。对于CBMN测定，在 体外 用0.5、1和2 Gy的剂量照射血培养物，以评估冷冻保存的全血样本淋巴细胞中的MN反应。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>15 mL centrifuge tubes</td>
			<td>Greiner</td>
			<td>188271</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well cell suspension plate&#160;</td>
			<td>VWR</td>
			<td>734-2779</td>
			<td></td>
		</tr>
		<tr>
			<td>96% alcohol</td>
			<td>ChemLab</td>
			<td>CL00.1807.2500</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic acid</td>
			<td>Merck life science</td>
			<td>8,18,75,52,500</td>
			<td></td>
		</tr>
		<tr>
			<td>Acridine orange</td>
			<td>Merck life science</td>
			<td>235474-5g</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Merck life science</td>
			<td>C5670-100g</td>
			<td></td>
		</tr>
		<tr>
			<td>cover slips</td>
			<td>VWR</td>
			<td>631-1365</td>
			<td>22 x 50</td>
		</tr>
		<tr>
			<td>Cryobox (Mr.Frosty)</td>
			<td>Nalgene, Sigma Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cryovials 2ml</td>
			<td>Novolab</td>
			<td>A04573</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytochalsin B&#160;</td>
			<td>Merck life science</td>
			<td>C6762-10</td>
			<td>10 mg</td>
		</tr>
		<tr>
			<td>Dimethyl sulphoxide (DMSO)</td>
			<td>Merck life science</td>
			<td>D4540-500ml</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal calf serum (FCS)</td>
			<td>Thermo Fischer scientific</td>
			<td>10270-106</td>
			<td></td>
		</tr>
		<tr>
			<td>Fixative 1</td>
			<td></td>
			<td></td>
			<td>Methanol/acetic acid/ringer in a ratio of 4:1:5</td>
		</tr>
		<tr>
			<td>Fixative 2</td>
			<td></td>
			<td></td>
			<td>Methanol/acetic acid in a ratio of 4:1</td>
		</tr>
		<tr>
			<td>GURR buffer</td>
			<td>Thermo Fischer scientific</td>
			<td>10582013</td>
			<td>phosphate buffer (pH 6.8)</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Merck life science</td>
			<td>1,04,93,60,250</td>
			<td>75 mM</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Merck life science</td>
			<td>1,04,87,30,250</td>
			<td></td>
		</tr>
		<tr>
			<td>Li-heparin tubes</td>
			<td>BD Life sciences</td>
			<td>367526-LH170 I.U.</td>
			<td>BD Vacutainer</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>fisher scinetific</td>
			<td>M/4000/17</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2HPO2</td>
			<td>Merck life science</td>
			<td>10,65,80,500</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Merck life science</td>
			<td>S7653-1kg</td>
			<td></td>
		</tr>
		<tr>
			<td>Object slides</td>
			<td>VWR</td>
			<td>MENZAA00000112E04</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Thermo Fischer scientific</td>
			<td>15140-122</td>
			<td>10,000 U/mL + 10,000 &#181;g/mL</td>
		</tr>
		<tr>
			<td>Phytohemagglutinin (PHA-M)</td>
			<td>Thermo Fischer scientific</td>
			<td>10576-015</td>
			<td></td>
		</tr>
		<tr>
			<td>Ringer solution</td>
			<td></td>
			<td></td>
			<td>contains NaCl, KCl, CaCl2 dissolved in distilled water</td>
		</tr>
		<tr>
			<td>RPMI-1640</td>
			<td>Thermo Fischer scientific</td>
			<td>52400041</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicon rubber adhesive sealent</td>
			<td>collall</td>
			<td>CF-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>Thermo Fischer scientific</td>
			<td>11360039</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile warm PBS (37 &#176;C)</td>
			<td></td>
			<td></td>
			<td>contains NaCl,&#160; Na2HPO2, KH2PO4 dissolved in distilled water</td>
		</tr>
		<tr>
			<td>&#946;-mercaptoethanol</td>
			<td>Thermo Fischer scientific</td>
			<td>31350-010</td>
			<td></td>
		</tr>
	</tbody>
</table>

the micronucleus assay on cryopreserved whole blood

体外胞质分裂阻滞微核 （CBMN） 测定是一种广泛用于放射生物学研究、生物剂量学、遗传毒性研究和体外放射敏感性测试的技术。这种细胞遗传学方法基于检测双核细胞中的微核，这是由于细胞分裂过程中染色体片段滞后而产生的。新鲜全血样本是CBMN检测的首选样本类型。然而，使用新鲜血液样本的缺点包括采血后立即处理，以及无需额外血液采样即可进行的重复分析次数有限。
由于对新鲜血液样本的需求在后勤方面可能具有挑战性，因此对冷冻保存的全血样本进行CBMN检测将具有很大的优势，尤其是在大规模患者研究中。本文描述了一种冷冻全血样本并对这些冷冻血液样本进行CBMN测定的方案。来自健康志愿者的血液样本在不同的时间点被冷冻和解冻，然后接受修改后的微核测定方案。结果表明，这种优化的程序可以对冷冻血液样本进行CBMN测定。所描述的冷冻保存方案对于其他细胞遗传学测定和需要增殖淋巴细胞的各种功能测定也非常有用。

为了验证方案的可重复性，我们对 30 名年龄在 17 至 65 岁之间的健康志愿者的冷冻保存血液样本进行了 MN 测定。该组的平均年龄为35岁。冷冻保存时间从1周到154周不等。将全血细胞培养物暴露于不同辐射剂量（0.5、1 和 2 Gy）后，在显微镜下检查 1,000 BN 细胞中的微核产量。圆润的双核细胞（如 图2所示）表明已成功从冷冻保存的全血样本中提取出健康的活细胞。值得注意的是，在超低温（液氮）下长期储存后，淋巴细胞的辐射反应保持稳定。这一观察结果与新鲜血液样本的预期反应一致。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65855/65855fig02v2.jpg"> 图 2:从 1 年前冷冻保存的冷冻全血样本中提取的双核细胞中观察到的微核。 （A） 放大倍数 200 倍， （B） 放大倍数 400 倍;指向MN的箭头。 比例尺 = 50 μm。 <a href="https://www.jove.com/files/ftp_upload/65855/65855fig02large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
随着辐射剂量（0.5、1和2 Gy）的增加，观察到微核产量呈线性二次增加（表1 和 图3）。假辐照对照样品中的MN产量（0 Gy）代表了背景MN产量，主要是滞后染色体的结果。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>剂量 （Gy）</td>
			<td>0</td>
			<td>0.5</td>
			<td>1</td>
			<td>2</td>
		</tr><tr><td>平均</td>
			<td>18</td>
			<td>107</td>
			<td>247</td>
			<td>601</td>
		</tr><tr><td>标清</td>
			<td>10.8</td>
			<td>23.9</td>
			<td>53.3</td>
			<td>101.1</td>
		</tr><tr><td>简历 （%）</td>
			<td>59.9</td>
			<td>22.3</td>
			<td>21.6</td>
			<td>16.8</td>
		</tr><tr><td>范围</td>
			<td>5-41</td>
			<td>62-140</td>
			<td>139-324</td>
			<td>395-755</td>
		</tr></tbody></table>表 1:在 30 名健康供体的冷冻保存全血样本中观察到的变异范围和系数以及平均 MN 产量，表明个体间的变异性。 缩写:CV = 变异系数;SD = 标准偏差。
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65855/65855fig03.jpg"> 图 3:在 30 名供体的对照组和辐照冷冻保存的全血样本中观察到的微核产量。 冷冻保存期从 1 周到 154 周不等。散点图显示单个值。聚类中的线表示组的平均值±标准差。缩写:MN = 微核;BN = 双核。 <a href="https://www.jove.com/files/ftp_upload/65855/65855fig03large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
为了研究人MN产量的个体间变异，计算了变异系数（CV）（见 表1）。对于辐射诱导的 MN，所有剂量（0.5、1 和 2 Gy）的 CV < 均为 25%，这表明修改后的方案具有良好的可重复性。

Watch this Scientific Journal Video about Cryopreservation of Whole Blood Samples for High Throughput Analysis at JoVE.com

作者聚焦:用于高通量分析的全血样品冻存 

在这里，我们提出了一种优化的方案，用于对冷冻保存的全血样品进行胞质分裂阻滞微核测定。这种用于微核分析的全血冷冻保存优化方法是大规模取样和多中心研究的可靠技术，也可用于其他血液相关检测。

冷冻保存全血的微核检测

The in vitro cytokinesis-block micronucleus (CBMN) assay is a widely used technique in radiobiology research, biological dosimetry, genotoxicity studies, ...

我们的研究主要集中在检查电离辐射对健康组织的影响。我们正在研究癌症患者和DNA修复障碍患者的染色体放射敏感性。此外，我们还参与了生物不对称性研究，评估了暴露于电离辐射引起的DNA损伤。为了推进我们领域的研究，我们使用自动化显微系统进行高通量分析。新鲜全血样本是微核测定的首选样本类型，因为它很容易获得，对患者的不适感最小。然而，在时间限制下，立即处理的需要可能在物流上具有挑战性。全血样品的冷冻允许重复分析，而无需进一步进行额外取样。该程序是在大规模和多中心研究中储存血液样本的一种简单且省时的方法。全血冷冻保存方案不仅为微核测定提供了优势，而且为其他需要淋巴细胞增殖的细胞遗传学测定或功能测定提供了优势。

the-micronucleus-assay-on-cryopreserved-whole-blood

Research

JoVE Journal

Cancer Research

The Micronucleus Assay on Cryopreserved Whole Blood

3.5K 次观看.  Ghent University. 我们的研究主要集中在检查电离辐射对健康组织的影响。我们正在研究癌症患者和DNA修复障碍患者的染色体放射敏感性。此外，我们还参与了生物不对称性研究，评估了暴露于电离辐射引起的DNA损伤。为了推进我们领域的研究，我们使用自动化显微系统进行高通量分析。新鲜全血样本是微核测定的首选样本类型，因为它很容易获得，对患者的不适感最小。然而，在时间?...

视频: 作者聚焦:用于高通量分析的全血样品冻存 

Capture and Release of Viable Circulating Tumor Cells from Blood

We demonstrate a method for size based capture of viable circulating tumor cell (CTC) from whole blood, along with the release of these cells from chip for downstream analysis and/or culture. The strategy employs the use of a novel Parylene C membrane slot pore microfilter to capture CTC and a coating of poly (N-iso-propylacrylamide) (PIPAAm) for thermoresponsive viable release of the captured CTC. The capture of live cells is enabled by leveraging the design of a slot pore geometry with specific dimensions to reduce the shear stress typically associated with the filtration process. While the microfilter exhibits a high capture efficiency, the release of these cells is non-trivial. Typically, only a small percentage of cells are released when techniques such as reverse flow or cell scraping are used. The strong adhesion of these epithelial cancer cells to the Parylene C membrane is attributable to non-specific electrostatic interaction. To counteract this effect, we employed the use of PIPAAm coating and exploited its thermal responsive interfacial properties to release the cells from the filter. Blood is first filtered at room temperature. Below 32 &#176;C, PIPAAm is hydrophilic. Thereafter, the filter is placed in either culture media or a buffer maintained at 37 &#176;C, which results in the PIPAAm turning hydrophobic, and subsequently releasing the electrostatically bound cells.

A protocol to utilize a poly(N-iso-propylacrylamide) (PIPAAm) coated microfilter for effective capture and thermoresponsive release of viable circulating tumor cells (CTC) is presented. This method allows capture of CTC from patients&#39; blood and subsequent release of viable CTC for downstream off-chip culture, analyses and characterization.

捕获和血液中的活循环肿瘤细胞释放

We demonstrate a method for size based capture of viable circulating tumor cell (CTC) from whole blood, along with the release of these cells from chip ...

科研

癌症研究

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the context of a whole organism. Sophisticated techniques to generate genetic mosaics facilitate induction of visually marked, genetically defined clones surrounded by normal tissue. The clones can be analyzed through diverse molecular, cellular and omics approaches. This study describes how to generate fluorescently labeled clonal tumors of varying malignancy in the eye/antennal imaginal discs (EAD) of Drosophila larvae using the Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. It describes procedures how to recover the mosaic EAD and brain from the larvae and how to process them for simultaneous imaging of fluorescent transgenic reporters and antibody staining. To facilitate molecular characterization of the mosaic tissue, we describe a protocol for isolation of total RNA from the EAD. The dissection procedure is suitable to recover EAD and brains from any larval stage. The fixation and staining protocol for imaginal discs works with a number of transgenic reporters and antibodies that recognize Drosophila proteins. The protocol for RNA isolation can be applied to various larval organs, whole larvae, and adult flies. Total RNA can be used for profiling of gene expression changes using candidate or genome-wide approaches. Finally, we detail a method for quantifying invasiveness of the clonal tumors. Although this method has limited use, its underlying concept is broadly applicable to other quantitative studies where cognitive bias must be avoided.

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

This protocol demonstrates how to generate fluorescently marked, genetically defined clonal tumors in the Drosophila eye/antennal imaginal discs (EAD). It describes how to dissect the EAD and brain from the third instar larvae and how to process them to visualize and quantify gene expression changes and tumor invasiveness.

该<em&gt;果蝇</em&gt;成虫盘瘤模型:基因的表达与肿瘤侵袭性的可视化和定量遗传马赛克

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the ...

Conditional Knockdown of Gene Expression in Cancer Cell Lines to Study the Recruitment of Monocytes/Macrophages to the Tumor Microenvironment

siRNA and shRNA-mediated knock down (KD) methods of regulating gene expression are invaluable tools for understanding gene and protein function. However, in the case that the KD of the protein of interest has a lethal effect on cells or the anticipated effect of the KD is time-dependent, unconditional KD methods are not appropriate. Conditional systems are more suitable in these cases and have been the subject of much interest. These include Ecdysone-inducible overexpression systems, Cytochrome P-450 induction system1, and the tetracycline regulated gene expression systems. 
The tetracycline regulated gene expression system enables reversible control over protein expression by induction of shRNA expression in the presence of tetracycline. In this protocol, we present an experimental design using functional Tet-ON system in human cancer cell lines for conditional regulation of gene expression. We then demonstrate the use of this system in the study of tumor cell-monocyte interaction.

This protocol serves as a scheme for setting up a functional Tet-ON system in cancer cell lines and its subsequent use, in particular for studying the role of tumor cell-derived proteins in recruitment of monocytes/macrophages to the tumor microenvironment.

肿瘤细胞系基因表达的条件性击倒研究单核/巨噬细胞在微环境中的招募

siRNA and shRNA-mediated knock down (KD) methods of regulating gene expression are invaluable tools for understanding gene and protein function. However, ...

Whole-body PET/MRI of Pediatric Patients: The Details That Matter

Integrated PET/MRI is a hybrid imaging technique enabling clinicians to acquire diagnostic images for tumor assessment and treatment monitoring with both high soft tissue contrast and added metabolic information. Integrated PET/MRI has shown to be valuable in the clinical setting and has many promising future applications. The protocol presented here will provide step-by-step instructions for the acquisition of whole-body 2-deoxy-2-(18F)fluoro-D-glucose (18F-FDG) PET/MRI data in children with cancer. It also provides instructions on how to combine a whole-body staging scan with a local tumor scan for evaluation of the primary tumor. The focus of this protocol is to be both comprehensive and time-efficient, which are two ubiquitous needs for clinical applications. This protocol was originally developed for children above 6 years, or old enough to comply with breath-hold instructions, but can also be applied to patients under general anesthesia. Similarly, this protocol can be modified to fit institutional preferences in terms of choice of MRI pulse sequences for both the whole-body scan and local tumor assessment.

This article provides comprehensive step-by-step instructions for the acquisition of whole-body 2-deoxy-2-(18F)fluoro-D-glucose (18F-FDG) PET/MRI scans for cancer staging of pediatric patients. The protocol was developed for children above 6 years, or old enough to comply with breath-hold instructions, but can be used for general anesthesia patients as well.

儿科患者全身 PET/MRI: 物质的细节

Integrated PET/MRI is a hybrid imaging technique enabling clinicians to acquire diagnostic images for tumor assessment and treatment monitoring with both ...

Methods for Evaluating the Role of c-Fos and Dusp1 in Oncogene Dependence

The demonstration of tyrosine kinase inhibitors (TKIs) in treating chronic myeloid leukemia (CML) has heralded a new era in cancer therapeutics. However, a small population of cells does not respond to TKI treatment, resulting in minimal residual disease (MRD); even the most potent TKIs fail to eradicate these cells. These MRD cells serve as a reservoir to develop resistance to therapy. Why TKI treatment is ineffective against MRD cells is not known. Growth factor signaling is implicated in supporting the survival of MRD cells during TKI treatment, but a mechanistic understanding is lacking. Recent studies demonstrated that an elevated c-Fos and Dusp1 expression as a result of convergent oncogenic and growth factor signaling in MRD cells mediate TKI resistance. The genetic and chemical inhibition of c-Fos and Dusp1 renders CML exquisitely sensitive to TKIs and cures CML in both genetic and humanized mouse models. We identified these target genes using multiple microarrays from TKI-sensitive and -resistant cells. Here, we provide methods for target validation using in vitro and in vivo mouse models. These methods can easily be applied to any target for genetic validation and therapeutic development.

Here, we describe protocols for the genetic and chemical validation of c-Fos and Dusp1 as a drug target in leukemia using in vitro and in vivo genetic and humanized mouse models. This method can be applied to any target for genetic validation and therapeutic development.

c-fos 和 dusp1 在癌基因依赖性中的作用评价方法

The demonstration of tyrosine kinase inhibitors (TKIs) in treating chronic myeloid leukemia (CML) has heralded a new era in cancer therapeutics. However, ...

Investigation of Genetic Dependencies Using CRISPR-Cas9-based Competition Assays

Gene perturbation studies have been extensively used to investigate the role of individual genes in AML pathogenesis. For achieving complete gene disruption, many of these studies have made use of complex gene knockout models. While these studies with knockout mice offer an elegant and time-tested system for investigating genotype-to-phenotype relationships, a rapid and scalable method for assessing candidate genes that play a role in AML cell proliferation or survival in AML models will help accelerate the parallel interrogation of multiple candidate genes. Recent advances in genome-editing technologies have dramatically enhanced our ability to perform genetic perturbations at an unprecedented scale. One such system of genome editing is the CRISPR-Cas9-based method that can be used to make rapid and efficacious alterations in the target cell genome. The ease and scalability of CRISPR/Cas9-mediated gene-deletion makes it one of the most attractive techniques for the interrogation of a large number of genes in phenotypic assays. Here, we present a simple assay using CRISPR/Cas9 mediated gene-disruption combined with high-throughput flow-cytometry-based competition assays to investigate the role of genes that may play an important role in the proliferation or survival of human and murine AML cell lines.

This manuscript describes a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) CRISPR-Cas9-based method for simple and expeditious investigation of the role of multiple candidate genes in Acute Myeloid Leukemia (AML) cell proliferation in parallel. This technique is scalable and can be applied in other cancer cell lines as well.

基于 crispr-cas9 的竞争检测对遗传依赖性的调查

Gene perturbation studies have been extensively used to investigate the role of individual genes in AML pathogenesis. For achieving complete gene ...

Flow Cytometric Analysis of Mitochondrial Reactive Oxygen Species in Murine Hematopoietic Stem and Progenitor Cells and MLL-AF9 Driven Leukemia

We present a flow cytometric approach for analyzing mitochondrial ROS in various live bone marrow (BM)-derived stem and progenitor cell populations from healthy mice as well as mice with AML driven by MLL-AF9. Specifically, we describe a two-step cell staining process, whereby healthy or leukemia BM cells are first stained with a fluorogenic dye that detects mitochondrial superoxides, followed by staining with fluorochrome-linked monoclonal antibodies that are used to distinguish various healthy and malignant hematopoietic progenitor populations. We also provide a strategy for acquiring and analyzing the samples by flow cytometry. The entire protocol can be carried out in a timeframe as short as 3-4 h. We also highlight the key variables to consider as well as the advantages and limitations of monitoring ROS production in the mitochondrial compartment of live hematopoietic and leukemia stem and progenitor subpopulations using fluorogenic dyes by flow cytometry. Furthermore, we present data that mitochondrial ROS abundance varies among distinct healthy HSPC sub-populations and leukemia progenitors and discuss the possible applications of this technique in hematologic research.

We describe a method for using multiparameter flow cytometry to detect mitochondrial reactive oxygen species (ROS) in murine healthy hematopoietic stem and progenitor cells (HSPCs) and leukemia cells from a mouse model of acute myeloid leukemia (AML) driven by MLL-AF9.

木尿造血干细胞和祖细胞和MLL-AF9驱动白血病线粒体反应氧物种的流细胞学分析

We present a flow cytometric approach for analyzing mitochondrial ROS in various live bone marrow (BM)-derived stem and progenitor cell populations from ...

Detection of Cell-Free DNA in Blood Plasma Samples of Cancer Patients

Identifying mutations in tumors of cancer patients is a very important step in disease management. These mutations serve as biomarkers for tumor diagnosis as well as for the treatment selection and its response in cancer patients. The current gold standard method for detecting tumor mutations involves a genetic test of tumor DNA by means of tumor biopsies. However, this invasive method is difficult to be performed repeatedly as a follow-up test of the tumor mutational repertoire. Liquid biopsy is a new and emerging technique for detecting tumor mutations as an easy-to-use and non-invasive biopsy approach.
Cancer cells multiply rapidly. In parallel, numerous cancer cells undergo apoptosis. Debris from these cells are released into a patient&#8217;s circulatory system, together with finely fragmented DNA pieces, called cell-free DNA (cfDNA) fragments, which carry tumor DNA mutations. Therefore, for identifying cfDNA based biomarkers using liquid biopsy technique, blood samples are collected from the cancer patients, followed by the separation of plasma and buffy coat. Next, plasma is processed for the isolation of cfDNA, and the respective buffy coat is processed for the isolation of a patient&#39;s genomic DNA. Both nucleic acid samples are then checked for their quantity and quality; and analyzed for mutations using next-generation sequencing (NGS) techniques.
In this manuscript, we present a detailed protocol for liquid biopsy, including blood collection, plasma, and buffy coat separation, cfDNA and germline DNA extraction, quantification of cfDNA or germline DNA, and cfDNA fragment enrichment analysis.

In this paper we present a detailed protocol for non-invasive liquid biopsy technique, including blood collection, plasma and buffy coat separation, cfDNA and germline DNA extraction, quantification of cfDNA or germline DNA, and cfDNA fragment enrichment analysis.

癌症患者血浆样本中无细胞DNA检测

Identifying mutations in tumors of cancer patients is a very important step in disease management. These mutations serve as biomarkers for tumor diagnosis ...

Mapping the Structure-Function Relationships of Disordered Oncogenic Transcription Factors Using Transcriptomic Analysis

Many cancers are characterized by chromosomal translocations which result in the expression of oncogenic fusion transcription factors. Typically, these proteins contain an intrinsically disordered domain (IDD) fused with the DNA-binding domain (DBD) of another protein and orchestrate widespread transcriptional changes to promote malignancy. These fusions are often the sole recurring genomic aberration in the cancers they cause, making them attractive therapeutic targets. However, targeting oncogenic transcription factors requires a better understanding of the mechanistic role that low-complexity, IDDs play in their function. The N-terminal domain of EWSR1 is an IDD involved in a variety of oncogenic fusion transcription factors, including EWS/FLI, EWS/ATF, and EWS/WT1. Here, we use RNA-sequencing to investigate the structural features of the EWS domain important for transcriptional function of EWS/FLI in Ewing sarcoma. First shRNA-mediated depletion of the endogenous fusion from Ewing sarcoma cells paired with ectopic expression of a variety of EWS-mutant constructs is performed. Then RNA-sequencing is used to analyze the transcriptomes of cells expressing these constructs to characterize the functional deficits associated with mutations in the EWS domain. By integrating the transcriptomic analyses with previously published information about EWS/FLI DNA binding motifs, and genomic localization, as well as functional assays for transforming ability, we were able to identify structural features of EWS/FLI important for oncogenesis and define a novel set of EWS/FLI target genes critical for Ewing sarcoma. This paper demonstrates the use of RNA-sequencing as a method to map the structure-function relationship of the intrinsically disordered domain of oncogenic transcription factors.

Intrinsically disordered domains are important for oncogenic fusion transcription factor function. To therapeutically target these proteins, a more detailed understanding of the regulatory mechanisms employed by these domains is required. Here, we use transcriptomics to map important structural features of the intrinsically disordered EWS domain in Ewing sarcoma.

使用转录组学分析绘制无序致癌转录因子的结构-功能关系

Many cancers are characterized by chromosomal translocations which result in the expression of oncogenic fusion transcription factors. Typically, these ...

An Automated Microscopic Scoring Method for the &#947;-H2AX Foci Assay in Human Peripheral Blood Lymphocytes

Ionizing radiation is a potent inducer of DNA damage and a well-documented carcinogen. Biological dosimetry comprises the detection of biological effects induced by exposure to ionizing radiation to make an individual dose assessment. This is pertinent in the framework of radiation emergencies, where health assessments and planning of clinical treatment for exposed victims are critical. Since DNA double strand breaks (DSB) are considered to be the most lethal form of radiation-induced DNA damage, this protocol presents a method to detect DNA DSB in blood samples. The methodology is based on the detection of a fluorescent labelled phosphorylated DNA repair protein, namely, &#947;-H2AX. The use of an automated microscopy platform to score the number of &#947;-H2AX foci per cell allows a standardized analysis with a significant decrease in the turn-around time. Therefore, the &#947;-H2AX assay has the potential to be one of the fastest and sensitive assays for biological dosimetry. In this protocol, whole blood samples from healthy adult volunteers will be processed and irradiated in vitro in order to illustrate the usage of the automated and sensitive &#947;-H2AX foci assay for biodosimetry applications. An automated slide scanning system and analysis platform with an integrated fluorescence microscope is used, which allows the fast, automatic scoring of DNA DSB with a reduced degree of bias.

This protocol presents the slide preparation and automated scoring of the &#947;-H2AX foci assay on peripheral blood lymphocytes. To illustrate the method and sensitivity of the assay, isolated lymphocytes were irradiated in vitro. This automated method of DNA DSB detection is useful for fast and high-throughput biological dosimetry applications.

一种人外周血淋巴细胞γ-H2AX病灶测定的自动显微镜评分方法

Ionizing radiation is a potent inducer of DNA damage and a well-documented carcinogen. Biological dosimetry comprises the detection of biological effects ...