Name: quantifying replication stress in ovarian cancer cells using single-stranded dna immunofluorescence
Uploaded: 2023-02-10T14:15:00
Description: Watch this Scientific Journal Video about Quantifying Replication Stress in Ovarian Cancer Cells Using Single-Stranded DNA Immunofluorescence at JoVE.com

PV得到了Alvin J. Siteman癌症中心通过巴恩斯犹太医院基金会的首届踏板事业资助，Marsha Rivkin卵巢癌研究中心的试点研究资助，Mary Kay Ash基金会和V基金会的癌症研究资助。NR得到了圣路易斯华盛顿大学NIH细胞和分子生物学培训T32资助的支持。

注意：卵巢癌细胞系OVCAR3用于这些步骤，但该协议广泛适用于多种其他细胞系，包括来自非卵巢来源的细胞系。该协议的原理图如图 2所示。
1. 电镀细胞
<ol><li>制作聚-L-赖氨酸涂层盖玻片。<ol><li>将高压灭菌的 12 mm 直径盖玻片加入含有聚-L-赖氨酸溶液的 50 mL 锥形管中，并放在摇臂上 15 分钟。</li>
		<li>在组织培养罩中吸出溶液。通过加入无...

<ol>
	<li>Tubbs, A., Nussenzweig, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endogenous+DNA+damage+as+a+source+of+genomic+instability+in+cancer.">Endogenous DNA damage as a source of genomic instability in cancer.</a> Cell. 168 (4), 644-656 (2017).</li><li>Konstantinopoulos, P. A., Ceccaldi, R., Shapiro, G. I., D'Andrea, 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=Homologous+recombination+deficiency:+Exploiting+the+fundamental+vulnerability+of+ovarian+cancer.">Homologous recombination deficiency: Exploiting the fundamental vulnerability of ovarian cancer.</a> Cancer Discovery. 5 (11), 1137-1154 (2015).</li><li>Gee, M. E., Faraahi, Z., McCormick, A., Edmondson, R. J. <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+repair+in+ovarian+cancer:+Unlocking+the+heterogeneity.">DNA damage repair in ovarian cancer: Unlocking the heterogeneity.</a> Journal of Ovarian Research. 11, 50 (2018).</li><li>Labidi-Galy, S. I., 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=High+grade+serous+ovarian+carcinomas+originate+in+the+fallopian+tube.">High grade serous ovarian carcinomas originate in the fallopian tube.</a> Nature communications. 8, 1093 (2017).</li><li>Kroeger, P. T., Drapkin, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenesis+and+heterogeneity+of+ovarian+cancer.">Pathogenesis and heterogeneity of ovarian cancer.</a> Current Opinion in Obstetrics &amp; Gynecology. 29 (1), 26-34 (2017).</li><li>Patch, A. -. 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=Whole-genome+characterization+of+chemoresistant+ovarian+cancer.">Whole-genome characterization of chemoresistant ovarian cancer.</a> Nature. 521 (7553), 489-494 (2015).</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=The+repertoire+of+mutational+signatures+in+human+cancer.">The repertoire of mutational signatures in human cancer.</a> Nature. 578 (7793), 94-101 (2020).</li><li>Byun, T. S., Pacek, M., Yee, M. -. C., Walter, J. C., Cimprich, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+uncoupling+of+MCM+helicase+and+DNA+polymerase+activities+activates+the+ATR-dependent+checkpoint.">Functional uncoupling of MCM helicase and DNA polymerase activities activates the ATR-dependent checkpoint.</a> Genes &amp; Development. 19 (9), 1040-1052 (2005).</li><li>Toledo, L. I., 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=ATR+prohibits+replication+catastrophe+by+preventing+global+exhaustion+of+RPA.">ATR prohibits replication catastrophe by preventing global exhaustion of RPA.</a> Cell. 155 (5), 1088-1103 (2013).</li><li>Brickner, J. R., Garzon, J. L., Cimprich, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Walking+a+tightrope:+The+complex+balancing+act+of+R-loops+in+genome+stability.">Walking a tightrope: The complex balancing act of R-loops in genome stability.</a> Molecular Cell. 82 (12), 2267-2297 (2022).</li><li>Cejka, P., Symington, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+end+resection:+Mechanism+and+control.">DNA end resection: Mechanism and control.</a> Annual Review of Genetics. 55, 285-307 (2021).</li><li>Kowalczykowski, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+overview+of+the+molecular+mechanisms+of+recombinational+DNA+repair.">An overview of the molecular mechanisms of recombinational DNA repair.</a> Cold Spring Harbor Perspectives in Biology. 7 (11), 016410 (2015).</li><li>Tirman, S., Cybulla, E., Quinet, A., Meroni, A., Vindigni, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PRIMPOL+ready,+set,+reprime.">PRIMPOL ready, set, reprime.</a> Critical Reviews in Biochemistry and Molecular Biology. 56 (1), 17-30 (2021).</li><li>Taglialatela, 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=REV1-Pol&#950;+maintains+the+viability+of+homologous+recombination-deficient+cancer+cells+through+mutagenic+repair+of+PRIMPOL-dependent+ssDNA+gaps.">REV1-Pol&#950; maintains the viability of homologous recombination-deficient cancer cells through mutagenic repair of PRIMPOL-dependent ssDNA gaps.</a> Molecular Cell. 81 (19), 4008-4025 (2021).</li><li>Cimprich, K. A., Cortez, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ATR:+An+essential+regulator+of+genome+integrity.">ATR: An essential regulator of genome integrity.</a> Nature Reviews. Molecular Cell Biology. 9 (8), 616-627 (2008).</li><li>Tirman, S., 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=Temporally+distinct+post-replicative+repair+mechanisms+fill+PRIMPOL-dependent+ssDNA+gaps+in+human+cells.">Temporally distinct post-replicative repair mechanisms fill PRIMPOL-dependent ssDNA gaps in human cells.</a> Molecular Cell. 81 (19), 4026-4040 (2021).</li><li>Cong, 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=Replication+gaps+are+a+key+determinant+of+PARP+inhibitor+synthetic+lethality+with+BRCA+deficiency.">Replication gaps are a key determinant of PARP inhibitor synthetic lethality with BRCA deficiency.</a> Molecular Cell. 81 (15), 3128-3144 (2021).</li><li>Xu, 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=CCNE1+copy+number+is+a+biomarker+for+response+to+combination+WEE1-ATR+inhibition+in+ovarian+and+endometrial+cancer+models.">CCNE1 copy number is a biomarker for response to combination WEE1-ATR inhibition in ovarian and endometrial cancer models.</a> Cell Reports. Medicine. 2 (9), 100394 (2021).</li><li>Konstantinopoulos, P. 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=A+Replication+stress+biomarker+is+associated+with+response+to+gemcitabine+versus+combined+gemcitabine+and+ATR+inhibitor+therapy+in+ovarian+cancer.">A Replication stress biomarker is associated with response to gemcitabine versus combined gemcitabine and ATR inhibitor therapy in ovarian cancer.</a> Nature Communications. 12, 5574 (2021).</li><li>Buisson, R., Boisvert, J. L., Benes, C. H., Zou, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+but+concerted+roles+of+ATR,+DNA-PK,+and+Chk1+in+countering+replication+stress+during+S+phase.">Distinct but concerted roles of ATR, DNA-PK, and Chk1 in countering replication stress during S phase.</a> Molecular Cell. 59 (6), 1011-1024 (2015).</li><li>Belan, 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=POLQ+seals+post-replicative+ssDNA+gaps+to+maintain+genome+stability+in+BRCA-deficient+cancer+cells.">POLQ seals post-replicative ssDNA gaps to maintain genome stability in BRCA-deficient cancer cells.</a> Molecular Cell. 82 (24), 4664-4680 (2022).</li><li>da Costa, A. A. B. 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=Targeting+replication+stress+in+cancer+therapy.">Targeting replication stress in cancer therapy.</a> Nature Reviews. Drug Discovery. 22 (1), 38-58 (2023).</li><li>Chen, R., Wold, 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=Replication+protein+A:+Single-stranded+DNA's+first+responder:+Dynamic+DNA-interactions+allow+replication+protein+A+to+direct+single-strand+DNA+intermediates+into+different+pathways+for+synthesis+or+repair.">Replication protein A: Single-stranded DNA's first responder: Dynamic DNA-interactions allow replication protein A to direct single-strand DNA intermediates into different pathways for synthesis or repair.</a> BioEssays. 36 (12), 1156-1161 (2014).</li><li>Bhat, K. P., Cortez, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RPA+and+RAD51:+Fork+reversal,+fork+protection,+and+genome+stability.">RPA and RAD51: Fork reversal, fork protection, and genome stability.</a> Nature Structural &amp; Molecular Biology. 25 (6), 446-453 (2018).</li><li>Mar&#233;chal, A., Zou, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RPA-coated+single-stranded+DNA+as+a+platform+for+post-translational+modifications+in+the+DNA+damage+response.">RPA-coated single-stranded DNA as a platform for post-translational modifications in the DNA damage response.</a> Cell Research. 25 (1), 9-23 (2015).</li></ol>

正如协议中提到的，包括一些实验对照以确保测定有效是有价值的。这些包括未经IdU处理的样品以及未经一抗处理的样品。两种阴性对照都应产生被DAPI染色但不含IdU信号的细胞。
根据实验条件和所使用的细胞系，可能需要不同的抗体稀释度才能获得最佳荧光信号。信号过多可能导致无法量化单个病灶，而信号太少可能意味着无法识别样品之间的实验差异。如果背景信号过多?...

基因组DNA经常受到来自各种内源性和外源性的多次攻击1。内源性损伤的频率与代谢副产物（如活性氧或醛）的水平直接相关，在包括卵巢癌在内的多种癌症类型中，代谢副产物本质上更高2，3。必须有效地解决DNA损伤;否则，它会促进遗传毒性病变，从而诱变。细胞修复遗传毒性病变的能力依赖于无差错DNA修复途径的功能以及响应DNA损伤的细胞周期进程的有效调节。值得注意的是，许多卵巢癌在p53中具有功能失活突变，因此具有有缺陷的G1 / S检查点，导致细胞启动DNA复制，尽管存在未修复的基因组病变4，5。超过50%的高级别浆液性卵巢癌（HGSOC）在BRCA1和BRCA2介导的同源重组（无差错DNA修复途径）中存在缺陷，并且约20%在基因CCNE1中具有扩增，从而过早地将G1细胞推入S期6，从而使卵巢癌的DNA损伤程度进一步复杂化。.内源性DNA损伤的高频率，有缺陷的检查点和故障的修复途径共同成倍地增强了卵巢癌中基因组病变的积累。这些病变可以阻碍关键细胞过程的进展，例如DNA复制和转录。如下所述，这些障碍催化细胞中单链DNA（ssDNA）的产生。
DNA的双螺旋对于保护基因组免受多种诱变过程的影响至关重要，例如自发脱嘌呤和去嘧啶，胞嘧啶脱氨酶的活性和氧化DNA损伤1，7。相比之下，ssDNA极易受到这些突变事件的影响。细胞中的多个过程可导致ssDNA的产生（图1）。其中包括以下内容：
（i）DNA复制机制的停滞：这导致DNA解旋酶和聚合酶解耦，留下ssDNA8，9的片段。
（ii）转录机制的停滞：RNA聚合酶的持续停滞导致产生称为R环的三链杂交DNA/RNA结构。R环形成将置换的，未转录的DNA暴露为单链10。
（iii）DNA末端切除：同源定向修复的开始需要产生3'ssDNA以催化寻找同源序列11。
（iv）D环：同源重组过程中的链侵入可导致非模板互补链的位移，从而产生ssDNA12。
（v）复制耦合间隙：在DNA复制过程中，滞后链合成以不连续的方式发生，首先产生冈崎片段，然后连接。处理冈崎片段的延迟或缺陷也可能导致ssDNA形成。最后，如果前导链上的复制叉遇到停滞的病变、DNA 聚合酶和灵长酶，PRIMPOL 可以在下游重新启动合成，从而在13，14 后面留下 ssDNA 间隙。
显然，这些事件中的大多数要么发生在DNA复制机制面临基因组病变时，要么发生在复制偶联修复期间，这表明更高的DNA损伤导致ssDNA水平增加。由于许多这些事件与复制相关，因此ssDNA的形成被认为是细胞15，16中“复制应激”的标志物。
在这里，我们描述了一种可用于可靠地定量细胞中ssDNA的测定方法。这种方法的简单性、可重复性和成本效益使其适合用于评估细胞中的复制应激反应。新兴研究表明，ssDNA的水平也可以预测对化疗的反应，例如PARP1 / 2酶，ATR和Wee1激酶17，18，19，20，21的抑制剂。这些抑制剂正在几种HGSOCs的治疗方案中进行研究22。因此，该测定也可以成为预测卵巢癌细胞化疗反应的有用工具。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>3% Paraformaldehyde (PFA)</td>
			<td>Fisher Scientific</td>
			<td>NC0179595</td>
			<td>10 g sucrose + 100 mL 10X PBS + water to make volume to 925 mL. Add 75 mL 40% Methanol free PFA, mix, and make aliquots of 50 mL before storage 
			Storage: Store in -20 &#176;C</td>
		</tr>
		<tr>
			<td>5-iodo-2&#39;-deoxyuridine (IdU)</td>
			<td>Sigma Aldrich</td>
			<td>I7125-5G</td>
			<td>MW = 354.10 g/mol.For 10 mM stock: dissolve 3.541 mg IdU to 1 mL 1 N liquid ammonia 
			Storage: Stored in -20 &#176;C</td>
		</tr>
		<tr>
			<td>Anti-BrdU antibody</td>
			<td>BD Biosciences</td>
			<td>347580</td>
			<td>Storage: Store in 4 &#176;C</td>
		</tr>
		<tr>
			<td>Anti-mouse Alexa Fluor Plus 488 secondary antibody</td>
			<td>Thermo Scientific</td>
			<td>A32766</td>
			<td>Light sensitive - keep in dark 
			Storage: Store in 4 &#176;C</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Sigma Aldrich</td>
			<td>A7906-100G</td>
			<td>Made by adding specific mass to volume of PBS 
			Storage: Store in 4 &#176;C</td>
		</tr>
		<tr>
			<td>Circular Cover Glass&#160;</td>
			<td>Electron Microscopy Sciences</td>
			<td>72230-01</td>
			<td></td>
		</tr>
		<tr>
			<td>NIS GA3 Software&#160;</td>
			<td>Nikon&#160;</td>
			<td>77010604</td>
			<td></td>
		</tr>
		<tr>
			<td>OVCAR3</td>
			<td>ATCC</td>
			<td>HTB-161</td>
			<td>Growth Media: RPMI supplemented with L-glutamine, 0.01 mg/mL bovine insulin; fetal bovine serum to a final concentration of 20% and 1X Pen Strep 
			Storage: Freezing Media: growth media + 5% DMSO and stored in -80 &#176;C</td>
		</tr>
		<tr>
			<td>Poly-L-Lysine solution</td>
			<td>Sigma Aldrich</td>
			<td>P4832-50ML</td>
			<td>Storage: Store in 4 &#176;C</td>
		</tr>
		<tr>
			<td>ProLong Diamond Antifade Mountant with DAPI</td>
			<td>Thermo Scientific</td>
			<td>P36962</td>
			<td>Storage: Store in 4 &#176;C</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA, 0.25%</td>
			<td>Genesee Scientific</td>
			<td>25-510</td>
			<td>Storage: Store in 4 &#176;C</td>
		</tr>
		<tr>
			<td>Water, sterile-filtered</td>
			<td>Sigma Aldrich</td>
			<td>W3500-6X500ML</td>
			<td>Storage: Store in 4 &#176;C</td>
		</tr>
	</tbody>
</table>

quantifying replication stress in ovarian cancer cells using single-stranded dna immunofluorescence

复制应激是几种卵巢癌的标志。复制应激可能来自多种来源，包括双链断裂、转录-复制冲突或扩增的癌基因，不可避免地导致单链 DNA （ssDNA） 的产生。因此，量化ssDNA为评估不同细胞类型和各种DNA损伤条件或治疗下的复制应激水平提供了机会。新出现的证据还表明，ssDNA可以预测对靶向DNA修复的化疗药物的反应。在这里，我们描述了一种详细的基于免疫荧光的方法来量化ssDNA。该方法涉及用胸苷类似物标记基因组，然后在非变性条件下基于抗体检测染色质的类似物。ssDNA的片段可以在荧光显微镜下可视化为病灶。病灶的数量和强度与细胞核中存在的ssDNA水平直接相关。我们还描述了一种用于量化ssDNA信号的自动化管道。该方法快速且可重复。此外，该方法的简单性使其适用于药物和遗传筛选等高通量应用。

代表性图像和来自未处理细胞和用0.5mM羟基脲处理24小时的细胞核的IdU病灶的定量如图 4所示。两个细胞核均在DAPI通道中染色并可识别。对这些图像的分析包括量化每个细胞核中的病灶数量。病灶的数量与复制应激的程度成正比。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64920/64920fig01.jpg"> 图1<str...

Watch this Scientific Journal Video about Quantifying Replication Stress in Ovarian Cancer Cells Using Single-Stranded DNA Immunofluorescence at JoVE.com

Quantifying Replication Stress in Ovarian Cancer Cells Using Single-Stranded DNA Immunofluorescence

在这里，我们描述了一种基于免疫荧光的方法来量化细胞中单链DNA的水平。这种高效且可重复的方法可用于检查复制应激，这是几种卵巢癌的共同特征。此外，该检测与自动分析管道兼容，进一步提高了其效率。

使用单链DNA免疫荧光量化卵巢癌细胞中的复制应激

Replication stress is a hallmark of several ovarian cancers. Replication stress can emerge from multiple sources, including double-strand breaks, ...

我们的协议提供了一种高效且易于重现的方法来量化单链DNA，单链DNA是各种细胞系中复制应激的标志物。此外，其简单性允许高通量应用和屏幕。特别是，这种方法允许快速量化复制应激，这是几种卵巢癌的标志。它还适用于主机自动分析软件，进一步提高效率。单链DNA现在正被积极考虑作为靶向DNA修复途径的化疗反应的生物标志物。因此，此方法非常适用于该方案。复制应激存在于卵巢癌背景或BRCA1，BRCA2缺陷癌症之外。因此，这种方法可以应用于各种其他细胞类型或疾病背景。首先，通过将高压灭菌的12毫米直径盖玻片和聚赖氨酸溶液添加到50毫升锥形管中来制作聚赖氨酸涂层的盖玻片，并放在摇臂上15分钟。在组织培养罩上吸出溶液。加入无菌水清洗盖玻片，然后将装有盖玻片的管子放回摇杆上五分钟。清洗盖玻片三次后，从管中吸出水并将盖玻片铺在无菌盘子上。吸出剩余的水，让它们在组织培养罩中干燥一小时或直到没有水滴残留。干燥后，用石蜡膜密封盘子并将其置于 4 摄氏度。为了防止细胞在预提取步骤中从盖玻片上脱落，在24孔板的每个孔中放置一个聚赖氨酸包被的盖玻片。一旦OVCAR3细胞达到70%至80%汇合度，从平板中吸出培养基并用5至7毫升PBS洗涤细胞。从平板中吸出PBS。吸出PBS后，加入1毫升0.25%胰蛋白酶，并将培养皿放入37摄氏度的培养箱中8至10分钟或直到细胞从板底部升起。用5至10毫升培养基收集细胞，并将细胞悬液加入锥形管中。使用标准程序用血细胞计数器手动计数细胞。接下来，稀释细胞悬液，并获得一个在三个群体后产生70%至80%汇合度的数字。在放置在24孔板孔中的聚赖氨酸盖玻片上加入1毫升细胞悬液，并在标准条件下在培养基中培养细胞。在一次群体加倍后，从平板中吸出现有培养基并用 10 微摩尔 IdU 脉冲细胞以进行随后的两次群体加倍。要收获细胞，请在冰上用冰冷的0.5%PBSTx替换培养基五分钟。为了固定细胞，吸出PBSTx并在室温下用3%多聚甲醛孵育细胞15分钟。用PBS洗涤三到四次后，将固定细胞保持在4摄氏度，直到进一步使用。固定后，使用0.5%PBSTx在冰上透化细胞五分钟，确保覆盖整个盖玻片。在室温下用1毫升0.2%PBST洗涤细胞三到四次后，吸出PBST并使用PBS制成的5%BSA在室温下封闭样品30分钟。对于免疫染色，通过将湿纸巾放在平底特百惠器皿上来准备统一的腔室。用石蜡膜盖住 24 孔板盖。将其放入加湿室中，然后将盖玻片放在板盖上。在盖玻片顶部加入60微升稀释的抗BrdU小鼠一抗。或者，为了减少抗体干燥并使用较小的体积，将一滴稀释的抗体放在封口膜上，然后将盖玻片翻转到其上。然后在 37 摄氏度下孵育一小时。孵育后，吸出一抗。将盖玻片放回24孔板中，并用0.2%PBST洗涤四次。如前所述，在盖玻片上加入稀释的二抗，并在室温下在黑暗中孵育一小时。标记显微镜载玻片，并使用DAPI安装介质将盖玻片安装在载玻片上。如果需要固化或硬化，请将载玻片在室温下在黑暗中存放 24 小时。来自未处理和用0.5毫摩尔羟基脲处理的细胞的细胞核在DAPI和IdU通道中染色和识别。对这些图像的分析包括量化每个细胞核中的病灶数量。病灶的数量与复制应激的程度成正比。重要的是要考虑所选细胞系的倍增时间，因为IdU脉冲的时间可能会因更长或更短的倍增期而变化。或者，我们可以探测细胞中的复制蛋白A以确认结果并评估细胞中的各种复制应激反应。该技术与任何DNA损伤诱导剂一起用于许多细胞系，以检查单株DNA的积累。因此，这种方法是广泛可访问和适用的。

Research

JoVE Journal

Cancer Research

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

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

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 ...

科研

癌症研究

Modified Terminal Restriction Fragment Analysis for Quantifying Telomere Length Using In-gel Hybridization

使用凝胶内杂交来定量端粒长度的修饰末端限制片段分析

There are several different techniques for measuring telomere length, each with their own advantages and disadvantages. The traditional approach, Telomere ...

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 ...

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 ...

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 ...

Semi-automatic PD-L1 Characterization and Enumeration of Circulating Tumor Cells from Non-small Cell Lung Cancer Patients by Immunofluorescence

免疫荧光对非小细胞肺癌患者循环肿瘤细胞的半自动PD-L1特征化及枚举

Circulating tumor cells (CTCs) derived from the primary tumor are shed into the bloodstream or lymphatic system. These rare cells (1&#8722;10 cells per mL ...

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

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 ...

Modeling the Effects of Hemodynamic Stress on Circulating Tumor Cells using a Syringe and Needle

使用注射器和针头模拟血液学应激对循环肿瘤细胞的影响

During metastasis, cancer cells from solid tissues, including epithelia, gain access to the lymphatic and hematogenous circulation where they are exposed ...

Generation and Culturing of High-Grade Serous Ovarian Cancer Patient-Derived Organoids

高级别浆液性卵巢癌患者来源类器官的生成和培养

Organoids are 3D dynamic tumor models that can be grown successfully from patient-derived ovarian tumor tissue, ascites, or pleural fluid and aid in the ...

Orthotopic Implantation of Patient-Derived Cancer Cells in Mice Recapitulates Advanced Colorectal Cancer

小鼠患者来源的癌细胞原位植入概括了晚期结直肠癌

Over the last decade, more sophisticated preclinical colorectal cancer (CRC) models have been established using patient-derived cancer cells and 3D ...