这项工作是由美国国立卫生研究院资助R00 CA160574（SFB）和由罗格斯大学生物技术培训计划（到SMM）的奖学金支持。

这个过程是经新泽西州的州立大学实验动物管理和使用委员会在罗格斯。小鼠按照美国国立卫生研究院指南实验动物护理和使用处理。科学家应咨询自己的国家和机构的动物组织建立和批准的准则。 开始之前，制备表1中列出的解决方案。 1，B细胞分离和激活安乐死采用的CO 2颈椎脱位鼠标。将鼠标这样身体的左侧是向上和喷鼠标，用70％的乙醇，直至毛发是湿的。解剖脾和地方35毫米组织培养皿用2ml洗涤缓冲液。在层流罩，用5毫升注射器的平头，轻轻的向上突破，在组织培养皿脾。 <ol><li>插入一个70微米的尼龙网成50毫升管和脾脏样品在2毫升的洗涤缓冲液转移到尼龙筛。轻轻地扰乱使用注射器的扁平端的脾脏中的网格。 </li><li>冲洗注射器的后面和35毫米的钢板用8ml的洗涤缓冲液，并通过70微米尼龙筛过滤。离心机在300×g离心在4℃下10分钟。 </li><li>吸出并弃去上清，重悬沉淀物在3ml的ACK裂解缓冲液中。移液器上下简要地破坏团块，并孵育5分钟。加入10毫升的洗涤缓冲液和离心分离机，在300×g离心在4℃下10分钟。 </li><li>吸出并弃去上清液，并通过在1ml的洗涤缓冲液轻敲试管底部，然后通过移液重悬沉淀。加入50μl的抗CD43 MACS微珠并在冰上孵育30分钟。加入10毫升的洗涤缓冲液，轻轻混匀，离心300×g离心4℃10分钟。加入10毫升的洗涤缓冲液，轻轻混匀，离心300×g离心4℃10分钟。 </li><li>设立迷你MACS柱放置一列上的MACS磁铁。定位标记的15毫升锥形管列如下。加入1毫升的洗涤缓冲液对柱，并允许向15ml试管中运行通过。 </li><li>吸去上清，重悬在1ml的洗涤缓冲液。加载样品在柱后的洗涤缓冲液中已运行通过。 </li><li>后的样品通过色谱柱运行，加入1 ml的洗涤缓冲液洗柱。加7毫升的洗涤缓冲液，直接向在15ml试管中收集的样品。 </li><li>计数使用血球细胞。传送/孔所需的500万细胞到50ml管细胞所需的体积。离心机在300×g离心在4℃下10分钟。补充B细胞中与LPS 1：500和IL-4 1：1,000。 </li><li>吸出上清液，并添加所需的百万个细胞/ ml的终浓度添加的B细胞培养基中的适当体积。 </li><li>板的细胞在6孔板中，用5ml的细胞在百万细胞/毫升，将其放入humidifi编细胞培养培养箱中于37℃，5％CO 2。 </li></ol> 2，B细胞固定<ol><li>加入50μl的秋水仙胺的对好含有5ml的B细胞（为100纳克/毫升的终浓度）孵育1小时，在37℃下。 Prewarm 75 mM的KCl溶液在37℃水浴。转移B细胞标记15ml试管中。用胶带和离心标签在300 XG，在4℃下10分钟。 </li><li>吸出上清液留1毫升的管中，通过移液重悬沉淀。加入5毫升预热氯化钾一滴一滴的同时点击或脉冲的旋涡。 </li><li>加入10 ml的KCl和通过反转混合。孵育在37℃水浴15分钟。加入5滴新鲜固定液和混合通过反转。离心机在300×g离心在4℃下10分钟。吸出上清液留1毫升的管，重悬沉淀用移液器上下吹吸。 </li><li>加入5毫升新鲜固定液一滴一滴的同时点击或脉冲在VORTEX。增加一个额外的10毫升固定液和温育30分钟，在室温下进行。 </li><li>离心机在300×g离心在4℃下10分钟。吸出上清液留1毫升管，重悬吹打。 ，增加14毫升新鲜固定液和离心机在300×g离心在4℃下10分钟。 </li><li>重复步骤2.7的两倍以上。 </li><li> ，增加14毫升新鲜固定液。密封瓶盖封口膜过夜存放于-20℃。 </li></ol> 3，制备中期染色体价差<ol><li>设定一个监管环境室22.9°C和52％的湿度。 </li><li>离心机在4℃下固定的B细胞，在300×g离心10分钟。吸出固定液留下70微升的管子，重悬吹打。 </li><li>放置在湿度箱标记的幻灯片以35°的角度。降35微升悬浮B细胞上一张幻灯片，每滴样品两张幻灯片。允许的幻灯片中的湿度室中干燥3​​0分钟，然后保存在幻灯片在37℃的幻灯片框。 </li></ol> 4，端粒PNA FISH <ol><li>探针的制备<ol><li>预温去离子甲酰胺至37℃。拌2微升端粒PNA探针与7微升去离子预热甲酰胺和孵育在37℃下振荡1小时。 </li><li>预温鱼预混至37℃。从步骤4.1.1添加7微升预热的鱼预混到混合物中，在37℃下进行1-3小时。 </li><li>变性的探针混合物8分钟，在80℃。在37℃下预退火的探针1小时。 </li></ol></li><li>染色体幻灯片的预处理<ol><li>预温热含有50ml的0.01M的HCl中和至37℃的水浴中载玻片染色缸。 </li><li>地方载玻片在含有2×SSC 5分钟，在室温下具有不同的载玻片染色缸。 </li><li>加入2微升蛋白酶库存到预热的载玻片染色缸和通过反转混合。传送滑动到该玻璃SLIDE-染色缸和孵化90秒，在37℃。 </li><li>洗涤载玻片2倍于含有1x PBS，每次5分钟，在室温下，振摇不同的载玻片染色缸。然后在室温下清洗载玻片5分钟，在1×PBS / MgCl 2的，摇动。 </li><li>传送的幻灯片到含有新鲜制备的1％甲醛/ 1X PBS / MgCl 2的，在室温下10分钟的载玻片染色缸。 </li><li>为5分钟，在含有1x PBS在室温下具有不同的载玻片染色缸洗载玻片，振摇。 </li><li>由具有含70％，90％和100％的乙醇分离载玻片染色罐制备乙醇脱水系列。 </li><li>传送滑动到每个罐3分钟，开始用70％乙醇，然后90％乙醇中，然后加入100％乙醇。完成后，将乙醇罐在-20℃。 </li><li>通过点击过量的乙醇到纸巾上，并以35度角支撑幻灯片允许的幻灯片风干。干燥后，马RK 18×18毫米面积上用金刚石笔幻灯片的后面杂交。 </li></ol></li><li>变性幻灯片鱼<ol><li>适用于120微升70％去离子甲酰胺/ 2×SSC为24 x 60毫米盖玻片。触摸滑动盖玻片。变性的滑动，在80℃的热板上进行90秒。 </li><li>很快，小心滑出盖玻片，并放置在冰冷的70％乙醇中的幻灯片为3分钟，接着进行90％乙醇和100％乙醇中各3分钟。使载玻片在空气中干燥。 </li><li>由衬塑料盒，密封的盖子，湿纸巾准备湿热试验箱。该文件应该是潮湿，但不饱和水。 </li><li>在潮湿的室内，申请步骤4.1.3预退火探针混合物来标记的区域在幻灯片上，盖上一个18×18毫米盖玻片，并在37℃1小时。 </li></ol></li><li>后孵化淘<ol><li>预温50％甲酰胺/ 2×SSC，1×SSC，以及4x SSC / 0.1％吐温20的45°;水浴。 </li><li>小心取出盖玻片，并在预热的50％甲酰胺/ 2×SSC 3xfor 5分钟洗载玻片每个在黑暗和摇晃。在预热的1×SSC中洗幻灯片3xfor每次5分钟，摇晃。最后，洗玻片在预热的4倍的SSC / 0.1％Tween-20的3xfor每次5分钟。 </li><li>染色玻片3分钟的DAPI在避光载玻片染色缸。 </li><li>洗幻灯片5分钟的2X SSC，颤抖。申请35微升的Mowiol安装介质，覆盖24×60毫米的盖玻片，并存储在幻灯片在4℃下。 </li></ol></li></ol>染色体的5微观分析<ol><li>分析使用的是标准的落射荧光显微镜中期幻灯片。使用一种成像平台与自动阶段如METASYSTEMS Metafer平台，以获得最佳效果。对于每个中期扩展，收集一个图像为DAPI荧光可视化的染色体，并用于从端粒P上的荧光信号的一个图像长袍。 （ 例如 ，Cy3标记的其他荧光剂是可用的，并同样很好地工作。） </li><li>用图像分析程序叠加图像，并分析。 </li></ol>

<ol>
	<li>Sancar, A., Lindsey-Boltz, L. A., Unsal-Kacmaz, K., Linn, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+of+mammalian+DNA+repair+and+the+DNA+damage+checkpoints.">Molecular mechanisms of mammalian DNA repair and the DNA damage checkpoints.</a> Ann Rev Biochem. 73, 39-85 (2004).</li><li>Sperka, T., Wang, J., Rudolph, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+damage+checkpoints+in+stem+cells,+ageing+and+cancer.">DNA damage checkpoints in stem cells, ageing and cancer.</a> Nature reviews Mol Cell Biol. 13 (9), 579-590 (2012).</li><li>Alexandrov, L. B., 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=Signatures+of+mutational+processes+in+human+cancer.">Signatures of mutational processes in human cancer.</a> Nature. 500 (7463), 415-421 (2013).</li><li>Bunting, S. F., 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=53BP1+inhibits+homologous+recombination+in+Brca1-deficient+cells+by+blocking+resection+of+DNA+breaks.">53BP1 inhibits homologous recombination in Brca1-deficient cells by blocking resection of DNA breaks.</a> Cell. 141 (2), 243-254 (2010).</li><li>Padilla-Nash, H. M., Barenboim-Stapleton, L., Difilippantonio, M. J., Ried, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectral+karyotyping+analysis+of+human+and+mouse+chromosomes.">Spectral karyotyping analysis of human and mouse chromosomes.</a> Nat Protoc. 1 (6), 3129-3142 (2006).</li><li>Callen, 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=Chimeric+IgH-TCRalpha/delta+translocations+in+T+lymphocytes+mediated+by+RAG.">Chimeric IgH-TCRalpha/delta translocations in T lymphocytes mediated by RAG.</a> Cell Cycle. 8 (15), 2408-2412 (2009).</li><li>Boboila, 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=Alternative+end-joining+catalyzes+robust+IgH+locus+deletions+and+translocations+in+the+combined+absence+of+ligase+4+and+Ku70.">Alternative end-joining catalyzes robust IgH locus deletions and translocations in the combined absence of ligase 4 and Ku70.</a> Natl Acad Sci USA. 107 (7), 3034-3039 (2010).</li><li>Benn, P., Delach, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+lymphocyte+culture+and+chromosome+analysis.">Human lymphocyte culture and chromosome analysis.</a> Cold Spring Harb Protoc. 2008, (2008).</li><li>Nguyen, H. N., Reijo Pera, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metaphase+spreads+and+spectral+karyotyping+of+human+embryonic+stem+cells.">Metaphase spreads and spectral karyotyping of human embryonic stem cells.</a> Cold Spring Harb Protoc. 2008, (2008).</li><li>Schreck, R. R., Disteche, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+4.">Chapter 4.</a> Chromosome banding techniques. Unit 2, (2001).</li><li>Gilley, D., 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-PKcs+is+critical+for+telomere+capping.">DNA-PKcs is critical for telomere capping.</a> Proc Natl Acad Sci USA. 98 (26), 15084-15088 (2001).</li><li>Bolzan, A. 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+aberrations+involving+telomeres+and+interstitial+telomeric+sequences.">Chromosomal aberrations involving telomeres and interstitial telomeric sequences.</a> Mutagenesis. 27 (1), 1-15 (2012).</li><li>Bolzan, A. D., Telomeres Bianchi, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=interstitial+telomeric+repeat+sequences+and+chromosomal+aberrations.">interstitial telomeric repeat sequences and chromosomal aberrations.</a> Mutat Res. 612 (3), 189-214 (2006).</li><li>Poon, S. S., Lansdorp, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+18.">Chapter 18.</a> Quantitative fluorescence in situ hybridization (Q-FISH). Unit 18 14, (2001).</li><li>Morales, J. 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=53BP1+and+p53+synergize+to+suppress+genomic+instability+and+lymphomagenesis.">53BP1 and p53 synergize to suppress genomic instability and lymphomagenesis.</a> Proc Natl Acad Sci USA. 103 (9), 3310-3315 (2006).</li><li>Halaby, M. 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=Synergistic+interaction+of+Rnf8+and+p53+in+the+protection+against+genomic+instability+and+tumorigenesis.">Synergistic interaction of Rnf8 and p53 in the protection against genomic instability and tumorigenesis.</a> PLoS Genet. 9 (1), e1003259 (2013).</li></ol>

作者没有利益冲突。

而活化的B淋巴细胞是特别适合于制备的有丝分裂染色体涂抹物，其它类型的细胞也可使用。 T淋巴细胞有许多共同的B细胞的优点，因为它们可以从脾或淋巴结进行纯化，并具有高的有丝分裂指数，当由生长在含有适当的促细胞分裂剂8中等刺激。是胚胎干细胞（ES细胞），也适合于中期染色体分析9。如果这些都无法使用，成纤维细胞可以使用，虽然用秋水仙胺更长的治疗（ 例?...

A correction was made to: Rapid Analysis of Chromosome Aberrations in Mouse B Lymphocytes by PNA-FISH.&#160;Table 1 (the list of solutions to prepare) was omitted from the Protocol section. The contents of Table 1 are as follows:
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Solution</td>
			<td>Reagents</td>
			<td>Comments</td>
			<td>Storage</td>
		</tr>
		<tr>
			<td rowspan="3">B Cell Wash Buffer</td>
			<td>500 ml 1x Hanks Balanced Salt Solution</td>
			<td rowspan="3">Filter through 0.22 &#956;m stericup filtration unit</td>
			<td rowspan="3">4 &#176;C</td>
		</tr>
		<tr>
			<td>5 ml 50 &#176;C heat inactivated FCS</td>
		</tr>
		<tr>
			<td>5 ml 100x Pen/Strep</td>
		</tr>
		<tr>
			<td rowspan="8">B Cell Medium</td>
			<td>418.5 ml RPMI-1640</td>
			<td rowspan="8">Filter through 0.22 &#956;m stericup filtration unit</td>
			<td rowspan="8">4 &#176;C</td>
		</tr>
		<tr>
			<td>50 ml 50 &#176;C heat inactivated FCS</td>
		</tr>
		<tr>
			<td>5 ml 100x Pen/Strep</td>
		</tr>
		<tr>
			<td>5 ml glutamine</td>
		</tr>
		<tr>
			<td>5 ml nonessential amino acids</td>
		</tr>
		<tr>
			<td>5 ml sodium pyruvate</td>
		</tr>
		<tr>
			<td>1.8 ml 14.2 M 2-mercaptoethanol</td>
		</tr>
		<tr>
			<td>5 ml 1M HEPES</td>
		</tr>
		<tr>
			<td>Fixative</td>
			<td>3:1 v/v methanol/glacial acetic acid</td>
			<td></td>
			<td>Make fresh</td>
		</tr>
		<tr>
			<td rowspan="2">0.075 M KCl</td>
			<td>1.395 g KCl</td>
			<td rowspan="2"></td>
			<td rowspan="2">Room Temperature</td>
		</tr>
		<tr>
			<td>250 ml distilled H2O</td>
		</tr>
		<tr>
			<td rowspan="3">FISH Master Mix</td>
			<td>40 ml 50% Dextran sulfate</td>
			<td rowspan="3">Vortex the solution and leave in a shaking platform overnight</td>
			<td rowspan="3">Aliquot and store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>20 ml 20x SSC</td>
		</tr>
		<tr>
			<td>40 ml sterile distilled H2O</td>
		</tr>
		<tr>
			<td rowspan="3">70% Formamide/2x SSC</td>
			<td>10 ml 20x SSC</td>
			<td rowspan="3">Adjust to pH 7 with 1M HCl</td>
			<td rowspan="3">Aliquot and store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>20 ml distilled H2O</td>
		</tr>
		<tr>
			<td>70 ml deionized formamide</td>
		</tr>
		<tr>
			<td rowspan="2">0.01 M HCl</td>
			<td>1 ml 1M HCl</td>
			<td rowspan="2">Adjust to pH 2.0</td>
			<td rowspan="2">Room Temperature</td>
		</tr>
		<tr>
			<td>99 ml distilled H2O</td>
		</tr>
		<tr>
			<td rowspan="2">1x PBS/MgCl2</td>
			<td>50 ml 1M MgCl2</td>
			<td rowspan="2"></td>
			<td rowspan="2">Room Temperature</td>
		</tr>
		<tr>
			<td>950 ml 1x PBS</td>
		</tr>
		<tr>
			<td rowspan="3">50% Formamide/2x SSC</td>
			<td>20 ml 20x SSC</td>
			<td rowspan="3">Adjust to pH 7.25 
			Formamide used in this step does not have to be deionized.</td>
			<td rowspan="3">Make fresh</td>
		</tr>
		<tr>
			<td>80 ml distilled H2O</td>
		</tr>
		<tr>
			<td>100 ml formamide</td>
		</tr>
		<tr>
			<td rowspan="3">4x SSC/0.1% Tween20</td>
			<td>50 ml 20x SSC</td>
			<td rowspan="3"></td>
			<td rowspan="3">Room Temperature</td>
		</tr>
		<tr>
			<td>200 ml distilled H2O</td>
		</tr>
		<tr>
			<td>250 &#956;l Tween20</td>
		</tr>
		<tr>
			<td rowspan="2">DAPI staining solution (80 ng/ml)</td>
			<td>40 &#956;l 0.2mg/ml DAPI stock</td>
			<td rowspan="2"></td>
			<td rowspan="2">4 &#176;C in light protected staining jar</td>
		</tr>
		<tr>
			<td>100 ml 2x SSC</td>
		</tr>
		<tr>
			<td rowspan="5">Mowiol Mounting Medium</td>
			<td>6 g glycerol</td>
			<td rowspan="5">Incubate at 50 &#176;C with stirring for 2hrs</td>
			<td rowspan="5">Aliquot and store at 4 &#176;C</td>
		</tr>
		<tr>
			<td>2.4 g Mowiol 4-88</td>
		</tr>
		<tr>
			<td>6 ml distilled H2O</td>
		</tr>
		<tr>
			<td>12 ml 0.2 M TrisCl pH 8.5</td>
		</tr>
		<tr>
			<td>230 &#956;l 1% Thimerosal (w/v in water)</td>
		</tr>
	</tbody>
</table>
Table 1. List of solutions.&#160;

A correction was made to: Rapid Analysis of Chromosome Aberrations in Mouse B Lymphocytes by PNA-FISH.

Erratum: Rapid Analysis of Chromosome Aberrations in Mouse B Lymphocytes by PNA-FISH

癌症是通过影响基因调节正常细胞生长的突变的积累而引起的。突变是变化所引起的DNA损伤的基因组的结构和序列的结果。 DNA损伤的发生原因，通过各种方法，包括外源试剂如电离辐射，和作为正常细胞的代谢，如通过与活性氧1接触存在的核苷酸碱基或损害自发脱氨的一种副产品。 虽然哺乳动物细胞中具有一系列的维修活动的可逆转的DNA损伤或恢复在断裂位点的序列，突变仍然积累在整个细胞的寿命。 DNA损伤可进一步促进衰老和干细胞的效能损失，其中两个是与衰老相关的疾病相关联的进程。在处理两个符号理解DNA损伤的修复，因此至关重要ificant问题的公众健康。越来越多的证据表明，哺乳动物DNA修复途径可以向肿瘤细胞的基因组3,4的演变，使得它更有必要了解参与抑制突变在分子水平上的处理。 染色体畸变的直接可视化是一个强大的和定量测定特定细胞类型的基因组不稳定性的程度手段。从细胞中冷凝的染色体在分裂中期，可以分离并利用光或荧光显微镜检查。这样的细胞遗传学方法已经在实践了几十年，并且可以用于证明易位或特定类型与DNA修复活性的损失相关联的染色体畸变的出现。该协议适合于一些潜在的扩展：染色体可以标记探针光谱核型分析（SKY）或多色荧光原位杂交（mFISH）确定易位5,6。这些技术也使染色体易位的频率和复杂的染色体易位结构来确定，这超出了能够与本协议提供的附加信息。备选地，序列特异性探针可产生和使用在选择的基因组位点7来测试DNA断裂的频率。 在这个协议中，我们描述了编制中期染色体由B淋巴细胞传播。荧光标记的肽核酸（PNA）探针，端粒重复序列的情况下，其有效标记的端粒在分裂中期染色体扩散该协议具有几个优点。 B细胞可以被诱导生长在高有丝分裂指数，使高品质的差可以持续进行制备。从转基因小鼠的B细胞也不太可能包含次要的基因突变，可以颠倒的贡献的分析特定基因的基因组的完整性。在PNA-FISH方法可以在一天内完成，并允许染色体断裂的更准确的得分。通过使用这种方法，特别是与在此协议中描述的特定设备的组合，所以能够产生非常一致的，高品质的差和快速分析的速率和基因组不稳定性的类型。

<table border="0" cellpadding="0" cellspacing="0" width="679">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Name</td>
			<td style="width:116px;">Company&#160;</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:176px;">&#160;Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">50% Dextran sulfate</td>
			<td>Intergen</td>
			<td>S4030</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Deionized formamide&#160;</td>
			<td>Ambion</td>
			<td>9342</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pepsin (5g)</td>
			<td>Sigma</td>
			<td>P 6887&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FCS</td>
			<td>Gemini</td>
			<td>100-106</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LPS (100mg)</td>
			<td>Sigma</td>
			<td>L2630</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">IL-4 (5&#956;g)</td>
			<td>Sigma</td>
			<td>I1020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PNA Telomere Probe</td>
			<td>PNA Bio Inc</td>
			<td>F1002</td>
			<td>(CCCTAACCCTAACCCTAA)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Colcemid</td>
			<td>Roche</td>
			<td>295892</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mowiol 4-88</td>
			<td>Sigma</td>
			<td>81381-50g</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CD43 (ly-48_) micro-beads</td>
			<td>Miltenyl Biotec.&#160;</td>
			<td>130-049-801</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DAPI</td>
			<td>Sigma</td>
			<td>32670-5MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Eclipse E800</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">AxioImager.Z2</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CDS-5 Cytogenetic Drying Chamber</td>
			<td>Thermotron</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

rapid analysis of chromosome aberrations in mouse b lymphocytes by pna-fish

有缺陷的DNA修复导致增加的基因组不稳定性，这是基因突变，导致肿瘤发生的根本原因。在不同类型的细胞的频率和染色体畸变的类型的分析，可以在DNA修复途径缺陷待阐明。理解哺乳动物DNA修复生物学已经由生产小鼠用在特定的基因敲除极大地帮助。此协议的目的是量化小鼠B淋巴细胞的基因组不稳定性。使用PNA-FISH探针（肽核酸-荧光原位杂交）的端粒的标签有利于基因组不稳定性的中期染色体利差的快速分析。 B细胞有相对的成纤维细胞特定的优势，因为他们有正常的染色体倍性和较高的有丝分裂指数。因此，B细胞的短期培养使基因组不稳定性的精确测量在原代细胞群这很可能具有较少的次级基因突变总比什么通常发现在转化的成纤维细胞或患者的细胞系。

从一个细胞衍生中期染色体应形成包含40条染色体（如果使用小鼠细胞）（ 图1A）的离散簇。每个染色体具有着丝点的一端，它是作为一个淡蓝色球体可见。在正常的染色体，2端粒信号被视为在染色单体和两个信号由每个着丝粒的末端。间期核将存在于滑动为好。这些将作为蓝色球体，用红色端粒信号，但是没有稠染色体（例如，参见图1B）中可见。在间期核中，可以忽?...

Watch this Scientific Journal Video about Rapid Analysis of Chromosome Aberrations in Mouse B Lymphocytes by PNA-FISH at JoVE.com

Rapid Analysis of Chromosome Aberrations in Mouse B Lymphocytes by PNA-FISH

DNA修复途径对于维持基因组的完整性，防止基因突变和癌症至关重要。该协议的目的是通过直接观察染色体畸变的量化基因组不稳定性在中期从原位杂交（FISH）对端粒DNA重复使用荧光小鼠B细胞的扩散。

在小鼠B淋巴细胞染色体畸变所PNA-FISH快速分析

Defective DNA repair leads to increased genomic instability, which is the root cause of mutations that lead to tumorigenesis. Analysis of the frequency ...

rapid-analysis-chromosome-aberrations-mouse-b-lymphocytes-pna

Research

JoVE Journal

Immunology and Infection

16.8K 次观看.  Rutgers, the State University of New Jersey.  DNA修复途径对于维持基因组的完整性，防止基因突变和癌症至关重要。该协议的目的是通过直接观察染色体畸变的量化基因组不稳定性在中期从原位杂交（FISH）对端粒DNA重复使用荧光小鼠B细胞的扩散。 

视频: Rapid Analysis of Chromosome Aberrations in Mouse B Lymphocytes by PNA-FISH

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in situ hybridization (FISH) for rapid culture-independent detection of Salmonella spp. Cell-charged tapes can also be placed face-down on selective agar for solid-phase enrichment prior to detection. Alternatively, low-volume liquid enrichments (liquid surface miniculture) can be performed on the surface of the tape in non-selective broth, followed by FISH and analysis via flow cytometry. To begin, sterile adhesive tape is brought into contact with fresh produce, gentle pressure is applied, and the tape is removed, physically extracting microbes present on these surfaces. Tapes are mounted sticky-side up onto glass microscope slides and the sampled cells are fixed with 10% formalin (30 min) and dehydrated using a graded ethanol series (50, 80, and 95%; 3 min each concentration). Next, cell-charged tapes are spotted with buffer containing a Salmonella-targeted DNA probe cocktail and hybridized for 15 - 30 min at 55&deg;C, followed by a brief rinse in a washing buffer to remove unbound probe. Adherent, FISH-labeled cells are then counterstained with the DNA dye 4',6-diamidino-2-phenylindole (DAPI) and results are viewed using fluorescence microscopy. For solid-phase enrichment, cell-charged tapes are placed face-down on a suitable selective agar surface and incubated to allow in situ growth of Salmonella microcolonies, followed by FISH and microscopy as described above. For liquid surface miniculture, cell-charged tapes are placed sticky side up and a silicone perfusion chamber is applied so that the tape and microscope slide form the bottom of a water-tight chamber into which a small volume (&le; 500 &mu;L) of Trypticase Soy Broth (TSB) is introduced. The inlet ports are sealed and the chambers are incubated at 35 - 37&deg;C, allowing growth-based amplification of tape-extracted microbes. Following incubation, inlet ports are unsealed, cells are detached and mixed with vigorous back and forth pipetting, harvested via centrifugation and fixed in 10% neutral buffered formalin. Finally, samples are hybridized and examined via flow cytometry to reveal the presence of Salmonella spp. As described here, our "tape-FISH" approach can provide simple and rapid sampling and detection of Salmonella on tomato surfaces. We have also used this approach for sampling other types of fresh produce, including spinach and jalape&ntilde;o peppers.

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple adhesive-tape-based approach for sampling of tomato and other fresh produce surfaces, followed by rapid whole cell detection of Salmonella using fluorescence in situ hybridization (FISH).

胶粘剂基于磁带的采样和荧光组合原位杂交快速检测沙门氏菌新鲜农产品

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in ...

科研

免疫与感染

Methods to Assess Beta Cell Death Mediated by Cytotoxic T Lymphocytes

Type 1 diabetes (T1D) is a T cell mediated autoimmune disease. During the pathogenesis, patients become progressively more insulinopenic as 
insulin production is lost, presumably this results from the destruction of pancreatic beta cells by T cells. Understanding the mechanisms of beta cell death during 
the development of T1D will provide insights to generate an effective cure for this disease. Cell-mediated lymphocytotoxicity (CML) assays have historically used the 
radionuclide Chromium 51 (51Cr) to label target cells. These targets are then exposed to effector cells and the release of 51Cr from target 
cells is read as an indication of lymphocyte-mediated cell death. Inhibitors of cell death result in decreased release of 51Cr.
 As effector cells, we used an activated autoreactive clonal population of CD8+ Cytotoxic T lymphocytes (CTL) isolated from a mouse stock transgenic for both the alpha and beta chains of the AI4 T cell receptor (TCR). Activated AI4 T cells were co-cultured with 51Cr labeled target NIT cells for 16 hours, release of 51Cr was recorded to calculate specific lysis 
Mitochondria participate in many important physiological events, such as energy production, regulation of signaling transduction, and apoptosis. The study of beta cell mitochondrial functional changes during the development of T1D is a novel area of research. Using the mitochondrial membrane potential dye Tetramethyl Rhodamine Methyl Ester (TMRM) and confocal microscopic live cell imaging, we monitored mitochondrial membrane potential over time in the beta cell line NIT-1. For imaging studies, effector AI4 T cells were labeled with the fluorescent nuclear staining dye Picogreen. NIT-1 cells and T cells were co-cultured in chambered coverglass and mounted on the microscope stage equipped with a live cell chamber, controlled at 37&deg;C, with 5% CO2, and humidified. During these experiments images were taken of each cluster every 3 minutes for 400 minutes.
Over a course of 400 minutes, we observed the dissipation of mitochondrial membrane potential in NIT-1 cell clusters where AI4 T cells were attached. In the simultaneous control experiment where NIT-1 cells were co-cultured with MHC mis-matched human lymphocyte Jurkat cells, mitochondrial membrane potential remained intact. This technique can be used to observe real-time changes in mitochondrial membrane potential in cells under attack of cytotoxic lymphocytes, cytokines, or other cytotoxic reagents.

Cell-mediated lymphocytotoxicity (CML) assays can be used to test autoreactive responses and study mechanisms of cell death in vitro. However, using live-cell confocal microscopic imaging techniques with fluorescent dyes, the type and kinetics of cell death as well as the pathways utilized can be studied in greater detail.

β细胞死亡介导细胞毒性T淋巴细胞的方法来评估

Type 1 diabetes (T1D) is a T cell mediated autoimmune disease.  During the pathogenesis, patients become progressively more insulinopenic as 
insulin ...

Isolation of Lymphocytes from Mouse Genital Tract Mucosa

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and bacteria 1. Mucosae are also inductive sites in the host to generate immunity against pathogens, such as the Peyers patches in the intestinal tract and the nasal-associated lymphoreticular tissue in the respiratory tract. This unique feature brings mucosal immunity as a crucial player of the host defense system. Many studies have been focused on gastrointestinal and respiratory mucosal sites. However, there has been little investigation of reproductive mucosal sites. The genital tract mucosa is the primary infection site for sexually transmitted diseases (STD), including bacterial and viral infections. STDs are one of the most critical health challenges facing the world today. Centers for Disease Control and Prevention estimates that there are 19 million new infectious every year in the United States. STDs cost the U.S. health care system $17 billion every year 2, and cost individuals even more in immediate and life-long health consequences. In order to confront this challenge, a greater understanding of reproductive mucosal immunity is needed and isolating lymphocytes is an essential component of these studies. Here, we present a method to reproducibly isolate lymphocytes from murine female genital tracts for immunological studies that can be modified for adaption to other species. The method described below is based on one mouse.&#x2003;

An efficient way to isolate lymphocytes from mouse genital tract is described. This method takes advantage of enzyme digestion and Percoll gradient separation to allow efficient isolation. This technique is also adaptable to for use in other species

小鼠生殖道黏膜淋巴细胞分离

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and ...

Rapid and Robust Analysis of Cellular and Molecular Polarization Induced by Chemokine Signaling

Cells respond to chemokine stimulation by losing their round shape in a process called polarization, and by altering the subcellular localization of many proteins. Classic imaging techniques have been used to study these phenomena. However, they required the manual acquisition of many cells followed by time consuming quantification of the morphology and the co-localization of the staining of tens of cells. Here, a rapid and powerful method is described to study these phenomena on samples consisting of several thousands of cells using an imaging flow cytometry technology that combines the advantages of a microscope with those of a cytometer. Using T lymphocytes stimulated with CCL19 and staining for MHC Class I molecules and filamentous actin, a gating strategy is presented to measure simultaneously the degree of shape alterations and the extent of co-localization of markers that are affected by CCL19 signaling. Moreover, this gating strategy allowed us to observe the segregation of filamentous actin (at the front) and phosphorylated Ezrin-Radixin-Moesin (phospho-ERM) proteins (at the rear) in polarized T cells after CXCL12 stimulation. This technique was also useful to observe the blocking effect on polarization of two different elements: inhibition of actin polymerization by a pharmacological inhibitor and expression of mutants of the Par6/atypical PKC signaling pathway. Thus, evidence is shown that this technique is useful to analyze both morphological alterations and protein redistributions.

Chemokine signaling elicits marked alterations of cellular morphology and some important redistributions of intracellular proteins. Here, a rapid and detailed protocol is provided to study these events.

细胞与分子极化的快速和强大的分析趋化因子诱导信号

Cells respond to chemokine stimulation by losing their round shape in a process called polarization, and by altering the subcellular localization of many ...

Detection of True IgE-expressing Mouse B Lineage Cells

B lymphocyte immunoglobulin heavy chain (IgH) class switch recombination (CSR) is a process wherein initially expressed IgM switches to other IgH isotypes, such as IgA, IgE and IgG. Measurement of IgH CSR in vitro is a key method for the study of a number of biologic processes ranging from DNA recombination and repair to aspects of molecular and cellular immunology. In vitro CSR assay involves the flow cytometric measurement surface Ig expression on activated B cells. While measurement of IgA and IgG subclasses is straightforward, measurement of IgE by this method is problematic due to soluble IgE binding to Fc&#949;RII/CD23 expressed on the surface of activated B cells. Here we describe a unique procedure for accurate measurement of IgE-producing mouse B cells that have undergone CSR in culture. The method is based on trypsin-mediated cleavage of IgE-CD23 complexes on cell surfaces, allowing for detection of IgE-producing B lineage cells by cytoplasmic staining. This procedure offers a convenient solution for flow cytometric analysis of CSR to IgE.

In vitro analysis of class switch recombination in mice is challenging due to cytophilic IgE molecules bound to Fc receptors on the surface of B cells. We describe a method for IgE detection using trypsin-mediated cleavage of surface-bound IgE prior to fixation and permeabilization for cytoplasmic fluorescence staining.

检测真正的IgE的表达小鼠B​​系细胞

B lymphocyte immunoglobulin heavy chain (IgH) class switch recombination (CSR) is a process wherein initially expressed IgM switches to other IgH ...

A Simple and Rapid Protocol to Non-enzymatically Dissociate Fresh Human Tissues for the Analysis of Infiltrating Lymphocytes

The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted as an important hallmark of cancer. Our research on breast cancer focuses on the active role that tumor infiltrating lymphocytes play in tumor progression and patient outcome. Toward this goal, we developed a methodology for the rapid isolation of intact lymphoid cells from normal and abnormal tissues in an effort to evaluate them proximate to their native state. Homogenates prepared using a mechanical dissociator show both increased viability and cell recovery while preserving surface receptor expression compared to enzyme-digested tissues. Furthermore, enzymatic digestion of the remaining insoluble material did not recover additional CD45+ cells indicating that quantitative and qualitative measurements in the primary homogenate likely genuinely reflect infiltrating subpopulations in the tissue fragment. The lymphoid cells in these homogenates can be easily characterized using immunological (phenotype, proliferation, etc.) or molecular (DNA, RNA and/or protein) approaches. CD45+ cells can also be used for subpopulation purification, in vitro expansion or cryopreservation. An additional benefit of this approach is that the primary tissue supernatant from the homogenates can be used to characterize and compare cytokines, chemokines, immunoglobulins and antigens present in normal and malignant tissues. This protocol functions extremely well for human breast tissues and should be applicable to a wide variety of normal and abnormal tissues.

This protocol describes the rapid non-enzymatic dissociation of fresh human tissue fragments for qualitative and quantitative assessment of CD45+ cells (lymphocytes/leukocytes) present in various normal and malignant human tissues. Additionally, the supernatant obtained from the primary tissue homogenate can be collected and stored for further analysis or experimentation.

一种简单快速议定书非酶离解新鲜人组织的浸润淋巴细胞的分析

The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted ...

Efficient Isolation Protocol for B and T Lymphocytes from Human Palatine Tonsils

Tonsils form a part of the immune system providing the first line of defense against inhaled pathogens. Usually the term &#8220;tonsils&#8221; refers to the palatine tonsils situated at the lateral walls of the oral part of the pharynx. Surgically removed palatine tonsils provide a convenient accessible source of B and T lymphocytes to study the interplay between foreign pathogens and the host immune system. This video protocol describes the dissection and processing of surgically removed human palatine tonsils, followed by the isolation of the individual B and T cell populations from the same tissue sample. We present a method, which efficiently separates tonsillar B and T lymphocytes using an antibody-dependent affinity protocol. Further, we use the method to demonstrate that human adenovirus infects specifically the tonsillar T cell fraction. The established protocol is generally applicable to efficiently and rapidly isolate tonsillar B and T cell populations to study the role of different types of pathogens in tonsillar immune responses.

Palatine tonsils are a rich source of B and T lymphocytes. Here we provide an easy, efficient and rapid protocol to isolate B and T lymphocytes from human palatine tonsils. The method described has been specifically adapted for studies of the viral etiology of tonsil inflammation known as tonsillitis.

从人类腭扁桃体B和T淋巴细胞有效隔离协议

Tonsils form a part of the immune system providing the first line of defense against inhaled pathogens. Usually the term &#8220;tonsils&#8221; refers to ...

Rapid Molecular Detection and Differentiation of Influenza Viruses A and B

Influenza is a contagious respiratory illness caused by influenza viruses A and B in humans and causes a significant amount of morbidity and mortality every year. The Influenza A and B assay was the first CLIA-waived molecular rapid flu test available. The Influenza A and B test works by employing isothermal amplification with influenza-specific primers followed by target detection with molecular beacon probes. Here, the performance of the Influenza A and B assay on frozen, archived nasopharyngeal swab (NPS) specimens stored in viral transport medium (VTM) were compared to a respiratory panel assay.
The performance of the Influenza A and B assay was evaluated by comparing the results to the respiratory panel reference method. The sensitivity for total influenza virus A was 67.5% (95% CI (CI), 56.6-78.5) and the specificity was 86.9% (CI, 71.0-100). For influenza virus B testing, the sensitivity and specificity were 90.2% (CI, 68.5-100) and 98.8% (CI, 68.5-100), respectively.
This system has the advantage of a significantly shorter test time than any other currently available molecular assay and the simple, pipette-free procedure runs on a fully integrated, closed, small-footprint system. Overall, the Influenza A and B assay evaluated in this study has the potential to serve as a point-of-care rapid influenza diagnostic test.

We describe a rapid, molecular-based Influenza A and B assay. The Influenza assay detects each target within 15 min by employing isothermal amplification with influenza-specific primers followed by target detection with molecular beacon probes. The Influenza A and B assay is user-friendly and required minimal hands-on time to perform.

快速分子检测和流感病毒A和B的分化

Influenza is a contagious respiratory illness caused by influenza viruses A and B in humans and causes a significant amount of morbidity and mortality ...

Molecular Diffusion in Plasma Membranes of Primary Lymphocytes Measured by Fluorescence Correlation Spectroscopy

Fluorescence correlation spectroscopy (FCS) is a powerful technique for studying the diffusion of molecules within biological membranes with high spatial and temporal resolution. FCS can quantify the molecular concentration and diffusion coefficient of fluorescently labeled molecules in the cell membrane. This technique has the ability to explore the molecular diffusion characteristics of molecules in the plasma membrane of immune cells in steady state (i.e., without processes affecting the result during the actual measurement time). FCS is suitable for studying the diffusion of proteins that are expressed at levels typical for most endogenous proteins. Here, a straightforward and robust method to determine the diffusion rate of cell membrane proteins on primary lymphocytes is demonstrated. An effective way to perform measurements on antibody-stained live cells and commonly occurring observations after acquisition are described. The recent advancements in the development of photo-stable fluorescent dyes can be utilized by conjugating the antibodies of interest to appropriate dyes that do not bleach extensively during the measurements. Additionally, this allows for the detection of slowly diffusing entities, which is a common feature of proteins expressed in cell membranes. The analysis procedure to extract molecular concentration and diffusion parameters from the generated autocorrelation curves is highlighted. In summary, a basic protocol for FCS measurements is provided; it can be followed by immunologists with an understanding of confocal microscopy but with no other previous experience of techniques for measuring dynamic parameters, such as molecular diffusion rates.

A method to measure protein diffusion in membranes of primary immune cells using fluorescence correlation spectroscopy (FCS) is described. In this paper, the use of antibodies for fluorescent labeling is illustrated.

由荧光相关光谱测量的一次淋巴细胞的细胞膜分子扩散

Fluorescence correlation spectroscopy (FCS) is a powerful technique for studying the diffusion of molecules within biological membranes with high spatial ...

Rapid Quantification of Mitogen-induced Blastogenesis in T Lymphocytes for Identifying Immunomodulatory Drugs

Lymphocyte proliferation in response to antigenic or mitogenic stimulation is a readily quantifiable phenomenon useful for testing immunomodulatory (i.e., immunosuppressive or immunostimulatory) chemical compounds and biologics. One of the earliest steps during mitogenesis is cell enlargement or blastogenic transformation, whereupon the cell volume increases before division. It is usually detectable in the first several hours of T-lymphocyte stimulation. Here, we describe a rapid method to quantify blastogenesis in T lymphocytes isolated from mouse spleens and human peripheral blood mononuclear cells (PBMCs) using an automated cell counter. Various commonly used proliferation assays for the most part are laborious and only reflect the overall population effect rather than individual cellular effects within a population. In contrast, the presented automated cell counter assay provides rapid, direct, and precise measurements of cell diameters that can be used for assessing the effectiveness of various mitogens and immunomodulatory drugs in vitro.

T-lymphocyte mitogenesis is accompanied by blastogenic transformation, whereupon the cell volume enlarges before cell division. Here, we describe a method to quantify blastogenesis in T lymphocytes using an automated cell counter with the capability of measuring cell diameters.

T淋巴细胞有丝分裂原诱导芽生的快速定量辨识免疫调节药物

Lymphocyte proliferation in response to antigenic or mitogenic stimulation is a readily quantifiable phenomenon useful for testing immunomodulatory (i.e., ...