Name: immunofluorescence imaging of dna damage and repair foci in human colon cancer cells
Uploaded: 2020-06-09T15:00:00
Description: Watch this Scientific Journal Video about Immunofluorescence Imaging of DNA Damage and Repair Foci in Human Colon Cancer Cells at JoVE.com

由中子/伽马辐射组成的中子混合束从波兰国家核研究中心的玛丽亚研究反应堆进入。K.M.O.得到波兰国家科学中心（Miniatura 2）第#2018/02/X/NZ5/02849号赠款的支持。

1. 细胞培养和实验的制备
<ol><li>维护人类结肠癌细胞，HCT-116，根据供应商的建议，在75厘米2培养瓶的单层含有10 mL的配制麦考伊的5a中法化，辅以10%的胎儿牛血清和1%的抗生素抗霉菌溶液。</li>
	<li>在37°C的加湿5%CO2环境中生长细胞，直到每2至3天进行一次中基更新的70%的汇合。</li>
	<li>获得BNCT光束（例如中子束加10BPA）和BNCT效应，将癌细胞作为重粒子杀死，并在?...

<ol>
	<li>Carter, R. 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=Complex+DNA+Damage+Induced+by+High+Linear+Energy+Transfer+Alpha-Particles+and+Protons+Triggers+a+Specific+Cellular+DNA+Damage+Response.">Complex DNA Damage Induced by High Linear Energy Transfer Alpha-Particles and Protons Triggers a Specific Cellular DNA Damage Response.</a> International Journal of Radiation Oncology, Biology, Physics. 100 (3), 776-784 (2018).</li><li>Kondo, 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=DNA+damage+induced+by+boron+neutron+capture+therapy+is+partially+repaired+by+DNA+ligase+IV.">DNA damage induced by boron neutron capture therapy is partially repaired by DNA ligase IV.</a> Radiation Environmental Biophysics. 55 (1), 89-94 (2016).</li><li>Sakai, W., Sugasawa, K. <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+Recognition+and+Repair+in+Mammalian+Global+Genome+Nucleotide+Excision+Repair.">DNA Damage Recognition and Repair in Mammalian Global Genome Nucleotide Excision Repair.</a> DNA Replication, Recombination, and Repair: Molecular Mechanisms and Pathology. , 155-174 (2016).</li><li>Rodriguez, 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=In+vitro+studies+of+DNA+damage+and+repair+mechanisms+induced+by+BNCT+in+a+poorly+differentiated+thyroid+carcinoma+cell+line.">In vitro studies of DNA damage and repair mechanisms induced by BNCT in a poorly differentiated thyroid carcinoma cell line.</a> Radiation Environmental Biophysics. 57 (2), 143-152 (2018).</li><li>Sollazzo, 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=Live+Dynamics+of+53BP1+Foci+Following+Simultaneous+Induction+of+Clustered+and+Dispersed+DNA+Damage+in+U2OS+Cells.">Live Dynamics of 53BP1 Foci Following Simultaneous Induction of Clustered and Dispersed DNA Damage in U2OS Cells.</a> International Journal of Molecular Sciences. 19 (2), (2018).</li><li>Jeggo, P., L&#246;brich, M. <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+DNA+damage+responses.">Radiation-induced DNA damage responses.</a> Radiation Protection Dosimetry. 122 (1-4), 124-127 (2006).</li><li>Sigurdsson, S., Van Komen, S., Petukhova, G., Sung, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Homologous+DNA+pairing+by+human+recombination+factors+Rad51+and+Rad54.">Homologous DNA pairing by human recombination factors Rad51 and Rad54.</a> The Journal of Biological Chemistry. 277 (45), 42790-42794 (2002).</li><li>Choi, E. H., Yoon, S., Hahn, Y., Kim, K. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+Dynamics+of+Rad51+and+Rad54+in+Response+to+Postreplicative+Stress+and+DNA+Damage+in+HeLa+Cells.">Cellular Dynamics of Rad51 and Rad54 in Response to Postreplicative Stress and DNA Damage in HeLa Cells.</a> Molecules and Cells. 40 (2), 143-150 (2017).</li><li>Burma, S., Chen, B. P., Murphy, M., Kurimasa, A., Chen, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ATM+phosphorylates+histone+H2AX+in+response+to+DNA+double-strand+breaks.">ATM phosphorylates histone H2AX in response to DNA double-strand breaks.</a> The Journal of Biological Chemistry. 276 (45), 42462-42467 (2001).</li><li>Nakamura, T. M., Du, L. L., Redon, C., Russell, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histone+H2A+phosphorylation+controls+Crb2+recruitment+at+DNA+breaks,+maintains+checkpoint+arrest,+and+influences+DNA+repair+in+fission+yeast.">Histone H2A phosphorylation controls Crb2 recruitment at DNA breaks, maintains checkpoint arrest, and influences DNA repair in fission yeast.</a> Molecular and Cellular Biology. 24 (14), 6215-6230 (2004).</li><li>Kuo, L. J., Yang, L. X. <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+-+a+novel+biomarker+for+DNA+double-strand+breaks.">Gamma-H2AX - a novel biomarker for DNA double-strand breaks.</a> In Vivo. 22 (3), 305-309 (2008).</li><li>Sage, E., Shikazono, N. <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+clustered+DNA+lesions:+Repair+and+mutagenesis.">Radiation-induced clustered DNA lesions: Repair and mutagenesis.</a> Free Radical Biology and Medicine. 107, 125-135 (2017).</li><li>Boguszewska, K., Szewczuk, M., Urbaniak, S., Karwowski, B. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review:+immunoassays+in+DNA+damage+and+instability+detection.">Review: immunoassays in DNA damage and instability detection.</a> Cellular and Molecular Life Sciences. 76 (23), 4689-4704 (2019).</li><li>M&#248;ller, 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=Potassium+bromate+as+positive+assay+control+for+the+Fpg-modified+comet+assay.">Potassium bromate as positive assay control for the Fpg-modified comet assay.</a> Mutagenesis. , (2020).</li><li>Sommer, S., Buraczewska, I., Kruszewski, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micronucleus+Assay:+The+State+of+Art,+and+Future+Directions.">Micronucleus Assay: The State of Art, and Future Directions.</a> International Journal of Molecular Sciences. 21 (4), 1534 (2020).</li><li>Bennett, B. T., Bewersdorf, J., Knight, 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=Immunofluorescence+imaging+of+DNA+damage+response+proteins:+optimizing+protocols+for+super-resolution+microscopy.">Immunofluorescence imaging of DNA damage response proteins: optimizing protocols for super-resolution microscopy.</a> Methods. 48 (1), 63-71 (2009).</li><li>Cheng, L., 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=Simultaneous+induction+of+dispersed+and+clustered+DNA+lesions+compromises+DNA+damage+response+in+human+peripheral+blood+lymphocytes.">Simultaneous induction of dispersed and clustered DNA lesions compromises DNA damage response in human peripheral blood lymphocytes.</a> PLoS One. 13 (10), 0204068 (2018).</li><li>Paull, T. T., 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=A+critical+role+for+histone+H2AX+in+recruitment+of+repair+factors+to+nuclear+foci+after+DNA+damage.">A critical role for histone H2AX in recruitment of repair factors to nuclear foci after DNA damage.</a> Current Biology. 10 (15), 886-895 (2000).</li><li>Okumura, K., 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=Relative+biological+effects+of+neutron+mixed-beam+irradiation+for+boron+neutron+capture+therapy+on+cell+survival+and+DNA+double-strand+breaks+in+cultured+mammalian+cells.">Relative biological effects of neutron mixed-beam irradiation for boron neutron capture therapy on cell survival and DNA double-strand breaks in cultured mammalian cells.</a> Journal of Radiation Research. 54 (1), 70-75 (2013).</li><li>Dagrosa, M. 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=First+evaluation+of+the+biologic+effectiveness+factors+of+boron+neutron+capture+therapy+(BNCT)+in+a+human+colon+carcinoma+cell+line.">First evaluation of the biologic effectiveness factors of boron neutron capture therapy (BNCT) in a human colon carcinoma cell line.</a> International Journal of Radiation Oncology, Biology, Physics. 79 (1), 262-268 (2011).</li><li>Dagrosa, 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=Studies+for+the+application+of+boron+neutron+capture+therapy+to+the+treatment+of+differentiated+thyroid+cancer.">Studies for the application of boron neutron capture therapy to the treatment of differentiated thyroid cancer.</a> Applied Radiation and Isotopes. 69 (12), 1752-1755 (2011).</li><li>Reynolds, 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=The+dynamics+of+Ku70/80+and+DNA-PKcs+at+DSBs+induced+by+ionizing+radiation+is+dependent+on+the+complexity+of+damage.">The dynamics of Ku70/80 and DNA-PKcs at DSBs induced by ionizing radiation is dependent on the complexity of damage.</a> Nucleic Acids Research. 40 (21), 10821-10831 (2012).</li><li>Rasche, L., 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=Analysis+of+Lymphocytic+DNA+Damage+in+Early+Multiple+Sclerosis+by+Automated+Gamma-H2AX+and+53BP1+Foci+Detection:+A+Case+Control+Study.">Analysis of Lymphocytic DNA Damage in Early Multiple Sclerosis by Automated Gamma-H2AX and 53BP1 Foci Detection: A Case Control Study.</a> PLoS One. 11 (1), 0147968 (2016).</li><li>Ban&#225;th, J. P., Macphail, S. H., 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=Radiation+sensitivity,+H2AX+phosphorylation,+and+kinetics+of+repair+of+DNA+strand+breaks+in+irradiated+cervical+cancer+cell+lines.">Radiation sensitivity, H2AX phosphorylation, and kinetics of repair of DNA strand breaks in irradiated cervical cancer cell lines.</a> Cancer Research. 64 (19), 7144-7149 (2004).</li><li>Barth, R. 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=Current+status+of+boron+neutron+capture+therapy+of+high+grade+gliomas+and+recurrent+head+and+neck+cancer.">Current status of boron neutron capture therapy of high grade gliomas and recurrent head and neck cancer.</a> Radiatiation Oncology. 7, 146 (2012).</li><li>Mirzaei, H. 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=Boron+neutron+capture+therapy:+Moving+toward+targeted+cancer+therapy.">Boron neutron capture therapy: Moving toward targeted cancer therapy.</a> Journal of Cancer Research and Therapeutics. 12 (2), 520-525 (2016).</li><li>Vitti, E. T., Parsons, J. 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+Radiobiological+Effects+of+Proton+Beam+Therapy:+Impact+on+DNA+Damage+and+Repair.">The Radiobiological Effects of Proton Beam Therapy: Impact on DNA Damage and Repair.</a> Cancers. 11 (7), 946 (2019).</li><li>Mohamad, O., 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=Carbon+Ion+Radiotherapy:+A+Review+of+Clinical+Experiences+and+Preclinical+Research,+with+an+Emphasis+on+DNA+Damage/Repair.">Carbon Ion Radiotherapy: A Review of Clinical Experiences and Preclinical Research, with an Emphasis on DNA Damage/Repair.</a> Cancers. 9 (6), 66 (2017).</li></ol>

α-H2AX和53BP1的源点、免疫化学染色频率常用于放射生物学，与DNA-DSB编号相关，被认为是监测DNA-DSB19诱导和修复的高效敏感标记。+-H2AX 和 53BP1 的共染色程序是检测 DNA-DSB 的标准程序。 α-H2AX foci 的形成与 53BP1 的招募有关，53BP1 是 DNA 损伤反应的调节器23。然而，不同的细胞系在 +-H2AX /53BP1 foci 11 的背景水平可能有所不同。已经证明，+-H2AX ...

在中子捕获治疗（BNCT）中使用的中子混合束激活的辐射诱导DNA损伤反应尚未完全确定。该协议提供了一个分步程序，通过免疫荧光染色（例如，在与中子混合束照射后在人类结肠癌细胞系中）检测修复蛋白的辐射诱导 foci （RIF）。
电离辐射（IR）诱发多种不同类型的DNA损伤（DNA-DSB，DNA-SSB，DNA碱损伤），其中DNA双链断裂是最基因毒性的DNA病变1。未修复的断点可导致细胞死亡，而失修的断发则增加了染色体重新排列、变异和失去决定性遗传信息的概率。对 DSB 作出反应的损伤响应途径包括DNA修复途径，如非同源端连接（NHEJ），一种需要Ku 70/80、DNA+PKcs、Xrcc4和DNA连结酶IV作为关键因子,,2、3、4、5、635的机制6。24在哺乳动物中，第二个主要DNA修复途径是同源重组修复（HRR）通路，需要关键成分——Rad52 epistasis基因家族——Rad51、Rad52、Rad54、Rad55、Rad57和Rad587。Rad51 和 Rad54 是与真核生物复制应力和 DNA 断裂相关的修复机制中涉及的关键人类重组因素。有趣的是，观察到HRR通路的降低调节增强了NHEJ通路，它容易出现错误，指向与基因组稳定性相关的HR通路8。
DSB形成的第一步是α-H2AX组蛋白的磷酸化在Ser-139由阿塔夏泰朗吉西亚突变激酶（ATM）从PI3激酶家族7,7，8。有趣的是，H2AX磷酸化可以通过免疫荧光技术很容易地可视化为foci（+-H2AX foci），使用特异性磷酸化H2AX6抗体。DSB 的数量和 +-H2AX foci 之间有 1：1 的关系，因此，DSB 标记 α-H2AX 通过对 foci 形成、大小和数量 6、7、8、9、10、116,7,8,9,10的特性进行了广泛的研究。α-H2AX foci的形成导致DNA损伤反应（DDR）蛋白和染色质改变因子的招募和积累，例如53BP1（p53结合蛋白1）、MDC1（DNA损伤检查点的中介）、BRCA1、Mre11/Rad50/Nbs1、PARP-1和许多其他修复因子，从而形成辐射诱导的 foci（RIF）。所有这些蛋白质通过直接或间接结合1、11、12与+-H2AX,11共同结合。
通过适当的敏感测试来检测DNA损伤和修复是十分重要的，因此，要更加重视高精确技术的发展。在DNA损伤和修复研究的背景下，该方法对于基因组DNA损伤的敏感检测、损伤类别描述以及DNA损伤和修复机制的量化至关重要。为了检测DNA受损的单细胞，彗星测定在放射生物学研究中是常用的。其他可用的细胞遗传学方法可识别染色体畸变，包括二心、易位、中心片段、环和色度型畸变，以及微核染色体损伤 （Mn）。放射生物学中最常用的方法是多中心染色体测定，因为它对辐射的特异性很高。例如，PCR，一种经典的分子方法，无法识别检测到的DNA损伤的类型。在这种情况下，免疫测量方法通过敏感性水平，因为反应是特异性的抗原和抗体之间。免疫荧光成像为不同蛋白质在不同叶层的出现提供了视觉证据，以响应DNA损伤剂，如电离辐射16。然而，通过实时PCR很容易检测到损伤和修复蛋白mRNA水平的激活水平，这是DNA损伤反应17背景下进一步分子研究的正确定量方法。
考虑到 +-H2AX foci 吸引修复因子 18，以监测细胞水平的 DNA 损伤和修复，我们根据对 NHEJ 通路 （DNA-PKcs） 和 HRR 通路 Rad52 代表性修复蛋白 foci 的分析，开发出可靠的免疫荧光染色程序。
在这里，我们建议使用这些蛋白质的免疫检测作为监测DNA-DSB诱导和修复的高效和敏感的程序。到目前为止，除了+-H2AX和53BP1标记19外，还没有关于DNA-DSB的数据，这些数据基于BNCT中子混合束照射后细胞水平上修复蛋白的临界。我们建议对HCT-116结肠癌细胞系进行适应，因为它本身富含DSB foci，作为DNA损伤分析的标准细胞系，因为RIF很容易检测。这种粘附细胞系易于维护，适合辐照程序。建议的程序基于大量与+-H2AX染色的一般免疫荧光程序相关的先前研究。然而，它包括有关选择适当的抗体与测试稀释每个代表性蛋白质属于每个修复路径的所有细节。此外，它描述了BNCT治疗中独特的中子混合光束的利用。然而，我们建议扩大研究与这两种方法，免疫荧光染色第一，然后，与高成本分子分析之前执行4,4，17。

<table>
	<tbody>
		<tr>
			<td>12 mm Coverslips</td>
			<td>VWR</td>
			<td>89015-725</td>
			<td></td>
		</tr>
		<tr>
			<td>35 mm Petri dishes</td>
			<td>Sarstedt</td>
			<td>7.183.390.000</td>
			<td></td>
		</tr>
		<tr>
			<td>4-Borono-L-phenylalanine</td>
			<td>SIGMA-ALDRICH</td>
			<td>17755</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic (100X)</td>
			<td>Gibco</td>
			<td>15240062</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-DNA PKcs (phospho S2056) antibody - ChIP Grad</td>
			<td>Abcam</td>
			<td>AB18192</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Mouse IgG (whole molecule)&#8211;FITC antibody produced in goat</td>
			<td>SIGMA-ALDRICH</td>
			<td>F0257</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-phospho-Histone H2A.X (Ser139) Antibody, clone JBW301</td>
			<td>MerckMillipore</td>
			<td>05-636</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-RAD52 antibody</td>
			<td>Abcam</td>
			<td>AB117097</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin Fraction V (BSA)</td>
			<td>Roche</td>
			<td>BSAV-RO</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI (4&#39;,6-Diamidino-2-Phenylindole, Dihydrochloride)</td>
			<td>ThermoFisher SCIENTIFIC</td>
			<td>D1306</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (Heat Inactivated)</td>
			<td>SIGMA-ALDRICH</td>
			<td>F9665</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat Anti-Rabbit IgG H&#38;L (Alexa Fluor 488)</td>
			<td>Abcam</td>
			<td>AB150077</td>
			<td></td>
		</tr>
		<tr>
			<td>HCT-116 cell line</td>
			<td>ATCC</td>
			<td>CCL-247&#8482;</td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>National Institute of Health (NIH)</td>
			<td>https://imagej.nih.gov/ij/</td>
			<td></td>
		</tr>
		<tr>
			<td>Image Pro</td>
			<td>Media cybernetics</td>
			<td>http://www.mediacy.com/imagepro</td>
			<td></td>
		</tr>
		<tr>
			<td>LUNA II Automated Cell Counter</td>
			<td>Logos Biosystems</td>
			<td>L40002</td>
			<td></td>
		</tr>
		<tr>
			<td>McCoy&#8217;s 5A Medium (Modified, with L-glutamine and sodium bicarbonate)</td>
			<td>SIGMA-ALDRICH</td>
			<td>M9309</td>
			<td></td>
		</tr>
		<tr>
			<td>microscope slides</td>
			<td>ThermoFisher SCIENTIFIC</td>
			<td>B-1198</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS)</td>
			<td>Hirszfeld Institute of Immunology and Experimental Therapy, PAS</td>
			<td>20.59.52.0</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>SIGMA-ALDRICH</td>
			<td>X100</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue Stain, 0.4%</td>
			<td>Logos Biosystems</td>
			<td>T13001</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA solution 0.25%</td>
			<td>SIGMA-ALDRICH</td>
			<td>T4049</td>
			<td></td>
		</tr>
	</tbody>
</table>

immunofluorescence imaging of dna damage and repair foci in human colon cancer cells

手稿的目的是提供一个逐步的协议，用于执行免疫荧光显微镜，以研究中子-伽马混合束在波龙中子捕获疗法（BNCT）中使用的辐射诱导DNA损伤反应。具体来说，建议的方法用于检测修复蛋白活化，可以使用DNA双链断裂（DNA-DSB）特有的抗体，将修复蛋白活化结果可视化为活体。在与中子混合束照射后，通过结肠癌细胞（HCT-116）的免疫荧光评估DNA修复源。DNA-DSB 是最基因毒性病变，在哺乳动物细胞中通过两种主要途径进行修复：非同源端连接通路 （NHEJ） 和同源重组修复 （HRR）。53BP1 与 DNA-DSB 编号相关，是放射生物学中常用标记物（如 +-H2AX）常用的，其免疫化学染色频率被认为是监测DNA-DSB诱导和修复的高效敏感标记。确定 +-H2AX foci 吸引修复蛋白，导致 DSB 附近的修复因子浓度更高。为了监测细胞水平的DNA损伤，计划对来自NHEJ通路和RAD52的DNA-PKcs代表性修复蛋白来源的存在进行免疫荧光分析。我们开发并引入了可靠的免疫荧光染色方案，用于检测辐射诱发的DNA损伤反应，其抗体具有NHEJ和HRR通路修复因子的特异性，并观察到辐射诱导的致病（RIF）。该方法可用于研究中子混合束辐射时高度激活的修复蛋白，从而表明修复途径的主导地位。

首先，我们分析了DNA-DSB、结肠癌细胞中的+H2AX foci、非辐射和中子混合束辐射的标准标记。+-H2AX foci 显示为不同的荧光点，并显示 DNA-DSB 的形成（因为每个 +-H2AX 荧光点表示单个 DSB）（参见图 1）。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61399/61399fig01.jpg"> 图1：细胞系HCT-116（无辐射）和中子混合光束照射后结肠癌细?...

Watch this Scientific Journal Video about Immunofluorescence Imaging of DNA Damage and Repair Foci in Human Colon Cancer Cells at JoVE.com

Immunofluorescence Imaging of DNA Damage and Repair Foci in Human Colon Cancer Cells

在中子捕获治疗（BNCT）中使用的中子混合束激活的辐射诱导DNA损伤反应尚未完全确定。该协议提供了一个分步程序，通过使用中子混合束照射后，通过人体结肠癌细胞系中的免疫荧光染色来检测修复蛋白质的辐射诱导 foci （RIF）。

人类结肠癌细胞DNA损伤与修复福西的免疫荧光成像

The purpose of the manuscript is to provide a step-by-step protocol for performing immunofluorescence microscopy to study the radiation-induced DNA damage ...

该协议提供了一个分步程序，通过与中子混合束照射后，通过人体结肠癌细胞系中的免疫荧光染色来检测修复蛋白质的辐射诱导源。免疫荧光成像是一种敏感方法，它为修复途径蛋白在 foci 中的外观提供视觉证据，以响应 DNA 损伤剂，如电离辐射。中子混合束用于中子捕获治疗BNCT，辐射引起的DNA损伤反应尚未完全确定。我们的协议也可用于分析细胞水平的生物效应，由于其他高 LET 束的辐射，如质子使用质子束治疗或使用强子疗法的碳离子。辐照后，从附着的细胞中取出介质，用2.5毫升PBS洗涤一次。然后用一毫升70%乙醇固定细胞10分钟。要渗透细胞，去除乙醇，用2.5毫升PBS清洗。在PBS中轻轻加入1毫升2%Triton X-100，覆盖盖玻片，并在室温下孵育细胞5分钟。用 PBS 洗涤细胞三次，用 PBS 中稀释的 2%BSA 的一毫升来阻断渗透。然后用2%BSA孵育细胞至少30分钟。要染色细胞，请按照文本手稿中的建议，加入 PBS 中稀释的正抗体的适当量。然后盖上培养皿，并放在一个塑料盒与保湿木质素，以保持湿度。在37摄氏度下孵育30分钟。孵育后，用PBS进行三次洗涤，并加入适当量的二级抗体。将盘子与保湿木质素一起回到塑料盒中，并在37摄氏度下孵育至少30分钟。然后用PBS重复洗涤，并添加100微升DAPI稀释到每毫升1微克的最终浓度，以对抗核。在室温下孵育细胞最多两分钟，然后用PBS重复洗涤。最后一次洗涤后，取出 PBS，轻轻将盖玻片放在安装介质的顶部，避免形成气泡。用指甲油密封盖玻片的边缘，在进行荧光显微镜之前，使安装介质变硬。Gamma-H2AX foci是DNA双链断裂的标准标记物，在中子混合、光束辐照和非辐照结肠癌细胞中被检测。foci 显示为明显的荧光点。在辐照细胞中观察到伽马-H2AX foci的直径较高。此外，还检测到高-LET阿尔法粒子的单个轨迹穿过核。免疫荧光显微镜用于检测代表性修复蛋白Rad52和DNA依赖蛋白激酶。与对照细胞相比，在照射后DNA断裂时测量了DNA依赖性蛋白激酶叶径的高均值。在辐照细胞中观察到聚类DNA双链断裂，其强度更高，为复杂、较大和聚类。免疫荧光染色最重要的阶段是细胞固定和细胞膜渗透。这些步骤是需要让抗体很容易渗透到这些细胞和细胞内修复蛋白质。此过程提供有关在细胞级别激活每个修复路径的信息。然后，使用实时PCR等测定方法，在分子水平上确认结果。这项技术使研究人员有可能探索辐射引起的DNA损伤反应，而对于使用抗癌疗法的不同光束，这种反应还没有完全确定。

Research

Education

JoVE Journal

JoVE Core

Cell Biology

Cancer Research

Unit 1

DNA Replication and Repair

SCIENTISTS IN ACTION

Evaluating In Vitro DNA Damage Using Comet Assay

Evaluating In Vitro DNA Damage Using Comet Assay

利用彗星试验评价体外DNA 损伤

DNA damage is a common phenomenon for each cell during its lifespan, and is defined as an alteration of the chemical structure of genomic DNA. Cancer ...

科研

癌症研究

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

Immunofluorescence Microscopy of ÃƒÆ’Ã…Â½Ãƒâ€šÃ‚Â³H2AX and 53BP1 for Analyzing the Formation and Repair of DNA Double-strand Breaks

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 ...

Characterization of Tumor Cells Using a Medical Wire for Capturing Circulating Tumor Cells: A 3D Approach Based on Immunofluorescence and DNA FISH

用医用金属丝捕获循环肿瘤细胞的肿瘤细胞: 基于免疫荧光和 DNA 的3D 方法

Circulating tumor cells (CTCs) are associated with poor survival in metastatic cancer. Their identification, phenotyping, and genotyping could lead to a ...

A Method to Define the Effects of Environmental Enrichment on Colon Microbiome Biodiversity in a Mouse Colon Tumor Model

一种确定环境富集对小鼠结肠肿瘤模型结肠菌群生物多样性影响的方法

Several recent studies have illustrated the beneficial effects of living in an enriched environment on improving human disease. In mice, environmental ...

Isolation of Human Endothelial Cells from Normal Colon and Colorectal Carcinoma - An Improved Protocol

正常结肠和结直肠癌患者内皮细胞的分离--一种改进的协议

Primary cells isolated from human carcinomas are valuable tools to identify pathogenic mechanisms contributing to disease development and progression. In ...

Characterizing DNA Repair Processes at Transient and Long-lasting Double-strand DNA Breaks by Immunofluorescence Microscopy

基于免疫荧光显微镜的瞬时和持久双链 dna 损伤 dna 修复过程的表征

The repair of double-stranded breaks (DSBs) in DNA is a highly coordinated process, necessitating the formation and resolution of multi-protein repair ...

In Vivo Immunofluorescence Localization for Assessment of Therapeutic and Diagnostic Antibody Biodistribution in Cancer Research

在Vivo免疫荧光定位中用于评估癌症研究中的治疗和诊断抗体生物分布

Monoclonal antibodies (mAbs) are important tools in cancer detection, diagnosis, and treatment. They are used to unravel the role of proteins in ...

In Vitro Evaluation of Oncogenic Transformation in Human Mammary Epithelial Cells

人类乳腺上皮细胞致癌转化的体外评价

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile ...

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

An Automated Microscopic Scoring Method for the ÃƒÅ½Ã‚Â³-H2AX Foci Assay in Human Peripheral Blood Lymphocytes

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 ...

Modeling Brain Metastasis by Internal Carotid Artery Injection of Cancer Cells

通过颈内动脉注射癌细胞模拟脑转移

Brain metastasis is a cause of severe morbidity and mortality in cancer patients. Critical aspects of metastatic diseases, such as the complex neural ...