Name: cell cycle-specific measurement of &#947;h2ax and apoptosis after genotoxic stress by flow cytometry
Uploaded: 2019-09-01T14:00:00
Description: Watch this Scientific Journal Video about Cell Cycle-specific Measurement of Î³H2AX and Apoptosis After Genotoxic Stress by Flow Cytometry at JoVE.com

我们感谢德国癌症研究中心（DKFZ）的流式细胞测定设施团队的支持。

1. 准备
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在任何类型的培养容器中制备 ±1 x 105细胞/样品作为起始材料。
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<li>例如，在将U87胶质细胞细胞暴露于电离辐射后进行时间过程实验：在每个时间点以三联成的T25瓶中辐照亚汇U87细胞。选择早期时间点（辐照后 15 分钟至 8 小时），跟随 DSB 修复动力学（+H2AX 级别）和后期时间点（24 小时至 96 小时），以评估残留 DSB 水平、细胞周期效应和凋亡。 注?...

<ol>
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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=Mesenchymal+stem+cells+retain+their+defining+stem+cell+characteristics+after+exposure+to+ionizing+radiation.">Mesenchymal stem cells retain their defining stem cell characteristics after exposure to ionizing radiation.</a> International Journal of Radiation Oncology Biology Physics. 87 (5), 1171-1178 (2013).</li><li>Nicolay, N. 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=Mesenchymal+stem+cells+are+resistant+to+carbon+ion+radiotherapy.">Mesenchymal stem cells are resistant to carbon ion radiotherapy.</a> Oncotarget. 6 (4), 2076-2087 (2015).</li><li>Nicolay, N. 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=Mesenchymal+stem+cells+exhibit+resistance+to+topoisomerase+inhibition.">Mesenchymal stem cells exhibit resistance to topoisomerase inhibition.</a> Cancer letters. 374 (1), 75-84 (2016).</li><li>Nicolay, N. 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=Mesenchymal+stem+cells+maintain+their+defining+stem+cell+characteristics+after+treatment+with+cisplatin.">Mesenchymal stem cells maintain their defining stem cell characteristics after treatment with cisplatin.</a> Scientific Reports. 6, 20035 (2016).</li><li>Nicolay, N. 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=Mesenchymal+stem+cells+are+sensitive+to+bleomycin+treatment.">Mesenchymal stem cells are sensitive to bleomycin treatment.</a> Scientific Reports. 6, 26645 (2016).</li><li>R&#252;hle, 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=Cisplatin+radiosensitizes+radioresistant+human+mesenchymal+stem+cells.">Cisplatin radiosensitizes radioresistant human mesenchymal stem cells.</a> Oncotarget. 8 (50), 87809-87820 (2017).</li><li>M&#252;nz, 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=Human+mesenchymal+stem+cells+lose+their+functional+properties+after+paclitaxel+treatment.">Human mesenchymal stem cells lose their functional properties after paclitaxel treatment.</a> Scientific Reports. 8 (1), 312 (2018).</li><li>R&#252;hle, 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=The+Radiation+Resistance+of+Human+Multipotent+Mesenchymal+Stromal+Cells+Is+Independent+of+Their+Tissue+of+Origin.">The Radiation Resistance of Human Multipotent Mesenchymal Stromal Cells Is Independent of Their Tissue of Origin.</a> International Journal of Radiation Oncology Biology Physics. 100 (5), 1259-1269 (2018).</li><li>Dean, P. N., Jett, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mathematical+analysis+of+DNA+distributions+derived+from+flow+microfluorometry.">Mathematical analysis of DNA distributions derived from flow microfluorometry.</a> Journal of Cell Biology. 60 (2), 523-527 (1974).</li><li>Fox, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+model+for+the+computer+analysis+of+synchronous+DNA+distributions+obtained+by+flow+cytometry.">A model for the computer analysis of synchronous DNA distributions obtained by flow cytometry.</a> Cytometry. 1 (1), 71-77 (1980).</li><li>Costes, S. V., Boissi&#232;re, A., Ravani, S., Romano, R., Parvin, B., Barcellos-Hoff, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+features+that+discriminate+between+foci+induced+by+high-+and+low-LET+radiation+in+human+fibroblasts.">Imaging features that discriminate between foci induced by high- and low-LET radiation in human fibroblasts.</a> Radiation Research. 165 (5), 505-515 (2006).</li><li>Meyer, B., Voss, K. -. O., Tobias, F., Jakob, B., Durante, M., Taucher-Scholz, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clustered+DNA+damage+induces+pan-nuclear+H2AX+phosphorylation+mediated+by+ATM+and+DNA-PK.">Clustered DNA damage induces pan-nuclear H2AX phosphorylation mediated by ATM and DNA-PK.</a> Nucleic Acids Research. 41 (12), 6109-6118 (2013).</li><li>Lopez Perez, 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=Superresolution+light+microscopy+shows+nanostructure+of+carbon+ion+radiation-induced+DNA+double-strand+break+repair+foci.">Superresolution light microscopy shows nanostructure of carbon ion radiation-induced DNA double-strand break repair foci.</a> FASEB. 30 (8), 2767-2776 (2016).</li><li>Schipler, A., Iliakis, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+double-strand-break+complexity+levels+and+their+possible+contributions+to+the+probability+for+error-prone+processing+and+repair+pathway+choice.">DNA double-strand-break complexity levels and their possible contributions to the probability for error-prone processing and repair pathway choice.</a> Nucleic Acids Research. 41 (16), 7589-7605 (2013).</li><li>Stenerlow, B., Hoglund, E., Carlsson, 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+fragmentation+by+charged+particle+tracks.">DNA fragmentation by charged particle tracks.</a> Advances in Space Research. 30 (4), 859-863 (2002).</li><li>Friedland, W., 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=Comprehensive+track-structure+based+evaluation+of+DNA+damage+by+light+ions+from+radiotherapy-relevant+energies+down+to+stopping.">Comprehensive track-structure based evaluation of DNA damage by light ions from radiotherapy-relevant energies down to stopping.</a> Scientific Reports. 7, 45161 (2017).</li><li>Pang, D., Chasovskikh, S., Rodgers, J. E., Dritschilo, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short+DNA+Fragments+Are+a+Hallmark+of+Heavy+Charged-Particle+Irradiation+and+May+Underlie+Their+Greater+Therapeutic+Efficacy.">Short DNA Fragments Are a Hallmark of Heavy Charged-Particle Irradiation and May Underlie Their Greater Therapeutic Efficacy.</a> Frontiers in Oncology. 6, 130 (2016).</li><li>B&#246;cker, W., Iliakis, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Computational+Methods+for+analysis+of+foci:+validation+for+radiation-induced+gamma-H2AX+foci+in+human+cells.">Computational Methods for analysis of foci: validation for radiation-induced gamma-H2AX foci in human cells.</a> Radiation Research. 165 (1), 113-124 (2006).</li><li>L&#246;brich, 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=&#947;H2AX+foci+analysis+for+monitoring+DNA+double-strand+break+repair:+Strengths,+limitations+and+optimization.">&#947;H2AX foci analysis for monitoring DNA double-strand break repair: Strengths, limitations and optimization.</a> Cell Cycle. 9 (4), 662-669 (2010).</li></ol>

该特色方法易于使用，并提供快速、准确和可重复的DNA损伤响应测量，包括双链断裂（DSB）诱导和修复、细胞周期效应和凋亡细胞死亡。与单个测定相比，这些端点的组合提供了更完整的两性关系图。该方法可广泛应用于放射生物学、治疗和保护领域的综合基因毒性测定，更普遍地应用于肿瘤学（例如，环境风险因素评估、药物筛选和基因评估）肿瘤细胞的不稳定性）。
该?...

肿瘤学的目标是在不损害正常细胞的情况下杀死或使癌细胞灭活。许多疗法要么直接或间接地诱导癌细胞中的基因毒性应激，但也在正常细胞中有些延伸。化疗或靶向药物通常与放射治疗相结合，以提高辐照肿瘤1、2、3、4、5的放射敏感性，从而减少辐射剂量，以尽量减少正常的组织损伤。
电离辐射和其他基因毒性剂会诱发不同类型的DNA损伤，包括基体修饰、链交联和单股或双链断裂。脱氧核糖核酸双链断裂（DSBs）是最严重的DNA病变，其诱导是电离辐射和各种细胞静血药物在放射化疗中细胞杀灭作用的关键。DSB不仅会损害基因组的完整性，而且促进突变6、7的形成。因此，不同的DSB修复途径，以及消除不可修复的细胞（如凋亡）的机制在进化过程中已经形成。整个DNA损伤反应（DDR）由复杂的信号通路网络调节，从DNA损伤识别和细胞周期抑制到允许DNA修复，到程序化细胞死亡或失活的情况下修复失败8。
已开发流式细胞测定法，用于在一次涵盖 DSB 感应和修复的综合测定中，对基因毒性应力后的 DDR 进行调查，以及修复失败的后果。它结合广泛应用的DSB标记βH2AX的测量与细胞周期和凋亡诱导的分析相结合，使用经典的subG1分析和更具体的卡巴塞-3活化评估。
将这些端点组合在一个测定中不仅减少了时间、人工和成本支出，还实现了对DSB感应和修复以及caspase-3活化的细胞周期特定测量。这种分析不可能与独立进行的分析，但它们对于全面了解基因毒性压力后DNA损伤反应具有高度相关性。许多抗癌药物，如细胞静态化合物，针对分裂细胞，其效率在很大程度上取决于细胞周期阶段。不同DSB修复过程的可用性也取决于细胞周期阶段和路径选择，这对修复精度至关重要，进而决定细胞9、10、11的命运。 12.此外，对DSB水平的细胞周期特定测量比集中分析更准确，因为DSB水平不仅取决于基因毒性化合物或辐射的剂量，还取决于细胞的DNA含量。
该方法已用于比较不同放射疗法的疗效，以克服胶质细胞瘤13的抗药性机制，并剖析电离辐射和骨肉瘤14、15中靶向药物的相互作用和非典型畸状类人横纹癌16。此外，所述方法已被广泛用于分析无线电和化疗对介体干细胞17、18、19、20、21的副作用。 22，23，24，这是修复治疗引起的正常组织损伤，并在再生医学中具有潜在的应用。

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			<td height="40" style="height:40px;width:405px;">1000 &#181;L filter tips</td>
			<td style="width:151px;">Nerbe plus</td>
			<td style="width:169px;">07-693-8300</td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="96">
			<td height="96" style="height:97px;">100-1000 &#181;L pipette</td>
			<td>Eppendorf</td>
			<td>3123000063</td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">12 x 75 mm Tubes with Cell Strainer Cap, 35 &#181;m mesh pore size</td>
			<td>BD Falcon</td>
			<td>352235</td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">15 mL tubes</td>
			<td>BD Falcon</td>
			<td>352096</td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">200 &#181;L filter tips</td>
			<td>Nerbe plus</td>
			<td>07-662-8300</td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">20-200 &#181;L pipette</td>
			<td>Eppendorf</td>
			<td>3123000055</td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">4&#8217;,6-Diamidin-2-phenylindol (DAPI)</td>
			<td>Sigma-Aldrich</td>
			<td>D9542</td>
			<td style="width:339px;">Dissolve in water at 200 &#181;g/ml and store aliquots at -20 &#176;C</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:405px;">Alexa Fluor 488 anti-H2A.X Phospho (Ser139) Antibody, RRID: AB_2248011</td>
			<td>BioLegend</td>
			<td>613406</td>
			<td style="width:339px;">Dilute 1:20</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:405px;">Alexa Fluor 647 Rabbit Anti-Active Caspase-3 Antibody, AB_1727414</td>
			<td>BD Pharmingen</td>
			<td>560626</td>
			<td style="width:339px;">Dilute 1:20</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">BD FACSClean solution</td>
			<td>BD Biosciences</td>
			<td>340345</td>
			<td style="width:339px;">For cytometer cleaning routine after measurement</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">BD FACSRinse solution</td>
			<td>BD Biosciences</td>
			<td>340346</td>
			<td style="width:339px;">For cytometer cleaning routine after measurement</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Dulbecco&#8217;s Phosphate Buffered Saline (PBS)</td>
			<td>Biochrom</td>
			<td>L 182</td>
			<td style="width:339px;">Dissolve in water to 1x concentration</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Dulbecco&#39;s Modified Eagle&#39;s Medium with stable glutamin</td>
			<td>Biochrom</td>
			<td>FG 0415</td>
			<td style="width:339px;">Routine cell culture material for the example cell line used in the protocol</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Ethanol absolute</td>
			<td>VWR</td>
			<td>20821.330</td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Excel software</td>
			<td>Microsoft</td>
			<td></td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">FBS Superior (fetal bovine serum)</td>
			<td>Biochrom</td>
			<td>S 0615</td>
			<td style="width:339px;">Routine cell culture material for the example cell line used in the protocol</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">FlowJo v10 software</td>
			<td>LLC</td>
			<td>online order</td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Fluoromount-G</td>
			<td>SouthernBiotech</td>
			<td>0100-01</td>
			<td style="width:339px;">Embedding medium for optional preparation of microscopic slides from stained samples</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">folded cellulose filters, grade 3hw</td>
			<td>NeoLab</td>
			<td>11416</td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">LSRII or LSRFortessa cytometer</td>
			<td>BD Biosciences</td>
			<td></td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">MG132</td>
			<td>Calbiochem</td>
			<td>474787</td>
			<td style="width:339px;">optional drug for apoptosis positive control</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Multifuge 3SR+</td>
			<td>Heraeus</td>
			<td></td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Paraformaldehyde</td>
			<td>AppliChem</td>
			<td>A3813</td>
			<td style="width:339px;">Prepare 4.5% solution fresh. Dilute in PBS by heating to 80 &#176;C with slow stirring under the fume hood. Cover the flask with aluminium foil to prevent heat loss. Let the solution cool to room temperature and adjust the final volume. Pass the solution through a cellulose filter.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:405px;">Phospho-Histone H3 (Ser10) (D2C8) XP Rabbit mAb (Alexa Fluor&#174; 555 Conjugate) RRID: AB_10694639</td>
			<td>Cell Signaling Technology</td>
			<td>#3475</td>
			<td style="width:339px;">Dilute 3:200</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">PIPETBOY acu 2</td>
			<td>Integra Biosciences</td>
			<td>155 016</td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Serological pipettes, 10 mL</td>
			<td>Corning</td>
			<td>4488</td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Serological pipettes, 25 mL</td>
			<td>Corning</td>
			<td>4489</td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Serological pipettes, 5 mL</td>
			<td>Corning</td>
			<td>4487</td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">SuperKillerTRAIL (modified TNF-related apoptosis-inducing ligand)</td>
			<td>Biomol</td>
			<td>AG-40T-0002-C020</td>
			<td style="width:339px;">optional drug for apoptosis positive control</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">T25 cell culture flasks</td>
			<td>Greiner bio-one</td>
			<td>690160</td>
			<td style="width:339px;">Routine cell culture material for the example cell line used in the protocol</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Trypsin/EDTA</td>
			<td>PAN Biotech</td>
			<td>P10-025500</td>
			<td style="width:339px;">Routine cell culture material for the example cell line used in the protocol</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">U87 MG glioblastoma cells</td>
			<td>ATCC</td>
			<td>ATCC-HTB-14</td>
			<td style="width:339px;">Example cell line used in the protocol</td>
		</tr>
	</tbody>
</table>

cell cycle-specific measurement of &#947;h2ax and apoptosis after genotoxic stress by flow cytometry

提出的方法或稍作修改的版本已经设计，以研究特定的治疗反应和副作用的各种抗癌治疗，用于临床肿瘤学。它能够定量和纵向分析基因毒性应激后DNA损伤反应，如放射治疗和多种抗癌药物引起的。该方法涵盖DNA损伤反应的所有阶段，为诱导和修复DNA双链断裂（DSBs）、细胞周期抑制和在修复失败时细胞凋亡导致细胞死亡提供终点。结合这些测量提供了有关细胞周期依赖治疗效果的信息，从而可以深入研究细胞增殖和应对DNA损伤的机制之间的相互作用。由于许多癌症治疗（包括化疗剂和电离辐射）的作用仅限于或根据特定的细胞周期阶段而产生强烈变化，相关分析依赖于一种可靠和可行的方法来评估治疗效果以细胞周期特定的方式在DNA上。单端点检测不可能实现这一点，而且该方法具有重要优势。该方法不局限于任何特定的细胞系，并已在众多肿瘤和正常组织细胞系中进行了彻底测试。除放射肿瘤学外，在肿瘤学的许多领域，包括环境危险因素评估、药物筛选和肿瘤细胞遗传不稳定性评价，可广泛应用。

人类U87或LN229胶质细胞细胞受到4Gy光子或碳离子电池辐射的照射。使用此处介绍的流式细胞测定方法（图3），在辐照后48小时的不同时间点测量细胞周期特异性βH2AX水平和凋亡。在两种细胞系中，碳离子诱导更高的βH2AX峰值水平，下降速度较慢，在24至48小时时与相同物理剂量的光子辐射相比显著升高（图4A）。这表明，与修复效率较低的光...

Watch this Scientific Journal Video about Cell Cycle-specific Measurement of Î³H2AX and Apoptosis After Genotoxic Stress by Flow Cytometry at JoVE.com

Cell Cycle-specific Measurement of &#947;H2AX and Apoptosis After Genotoxic Stress by Flow Cytometry

该方法结合了DNA双链断裂（DSB）、细胞循环分布和凋亡的定量分析，实现了对DSB诱导和修复以及修复失败后果的细胞周期特定评估。

细胞周期特异性测量βH2AX和细胞凋亡后热源毒性应力通过流细胞测定

The presented method or slightly modified versions have been devised to study specific treatment responses and side effects of various anti-cancer ...

该方法提供了有关细胞周期依赖治疗效果的信息，并允许和深入研究细胞增殖与应对DNA损伤的应对机制之间的相互作用。该方法将DNA损伤反应的重要终点，包括双链碎片识别和修复、细胞循环效应，以及最终细胞凋亡死亡，合并为一个全面的测定方法。我们主要利用这项技术研究临床肿瘤学中放射化学治疗的具体治疗反应和副作用。该测定对放射生物学和放射治疗领域的相关研究非常有用，但也可以应用于肿瘤学的其他各个领域。按照标准细胞收集程序从培养物中收集细胞后，将颗粒重新在PBS的一毫升中重新产生。将细胞转移到两毫升的固定溶液在引擎盖。在室温下孵育10分钟后，通过离心收集细胞，并蒸馏上经。将单元格固定为单个单元格悬浮液非常重要，并且所有样本的固定时间不变。给粘附细胞足够的时间分离，以获得很好的圆润细胞进行分析。然后通过敲击和重新暂停细胞在三毫升的70%乙醇中松开颗粒。要在染色前清洗细胞，通过离心沉淀细胞，然后通过敲击松开颗粒。然后用三毫升的洗涤溶液和一毫升的洗涤溶液重新暂停细胞。每次洗涤之间丢弃上一杯。最后一次洗涤后，小心吸进上经剂，而不干扰颗粒，并在感兴趣的抗体鸡尾酒的100微升中重新暂停 lucent 颗粒。在室温下一小时后，通过离心收集细胞，并小心吸上经剂，而不会干扰颗粒。然后，将松动的颗粒重新在 100 至 250 微升的 DNA 染色溶液中，并通过具有 35 微米网孔大小的样品管的细胞过滤器盖分配细胞。要通过流式细胞分析来分析细胞，请在圆度计窗口中打开参数选项卡，然后选择表中所示的参数。接下来，打开工作表窗口，并创建指示的绘图。将控制样品加载到圆度计上，然后按下运行按钮。在浏览器窗口中选择第一个管，然后单击管名称。在检查器窗口的参数选项卡中调整前向和侧散射和 DAPI 的检测器电压。按备用按钮继续软件中的工作表设置。使用多边形门工具定义向前散点区域中的单元格总体与侧散点区域图，并使用矩形门工具定义 DAPI 中的单个单元格总体与 DAPI 区域图。按控件 G 以显示总体层次结构，然后单击默认门名以重命名它们。随后，右键单击所有直方图，从上下文菜单中选择显示人口和单个单元格。获取控制和处理样品，以优化抗体耦合荧光的检测器电压，覆盖信号的全部动态范围。要测量样品，请按圆度仪上的运行按钮，并使用软件中的采集仪表板测量样品。然后选择文件、导出和实验以在对话框中选择目录导出以将数据另存为点 FCS 文件。要评估数据，请将点 FCS 文件拖入流式细胞测量分析软件的样本浏览器中。并使用多边形工具定义前进和侧散射区域图中的单元格总体，并排除分析中的碎片。在 DAPI 与 DAPI 区域图中，使用矩形工具定义单个单元格的填充，并排除分析中的任何细胞双精度或块。在DAPI区域直方图中，使用二角器工具将具有正常DNA含量的单个细胞与具有降解DNA的凋亡细胞进行区分。选择工具生物学和细胞周期打开细胞周期建模工具，并选择D dean-Jett Fox来估计G一个S和G两个M阶段的细胞频率。在 DAPI 中创建 G 一 S 和 G 二和 M 相门，与细胞周期总体的 DAPI 区域图，以实现特定于细胞周期的伽玛 H 两个 A X 测量。使用双子体工具在细胞周期群的Alexa 555区域直方图中分辨磷石H三个正细胞和负细胞，并区分单细胞群的Alexa 647区域直方图中的CAS基三个阳性和负细胞。按控件 T 打开表编辑器并按指示配置表。然后选择"要文件"以在菜单功能区的输出部分中设置格式和目标，然后将数据导出到传播工作表中。碳离子在胶质母细胞细胞中诱导更高的伽马H两个 AX 峰值水平，与同一物理剂量的光子辐射相比，这些细胞在下降较慢，并在 24 至 48 小时内保持显著升高。对于碳离子和光子辐射，伽马H两个 AX水平在G一相细胞中最高，可能是因为DNA双链断裂修复仅限于细胞周期的这个阶段非霍莫洛古斯结束连接的通路。与较高的双链断裂诱导率和较慢的修复动力学一起，碳离子在G两相中诱导更强、更持久的细胞周期抑制，并且比光子具有更高的凋亡率。根据治疗效果的强度，不同细胞周期相的分辨率可能具有挑战性。在某些细胞系中，DAPI浓度对细胞周期轮廓的质量有很大影响，需要进行调整。流式细胞学后，可以从剩余的样品中准备幻灯片，以评估伽马 H 两个 AX foci 的数量、大小和形状，并区分焦核和双核伽马 H 两个 AX 染色。利用我们的方法，我们可以证明，中层干细胞对电离辐射和托托莫美酶抑制具有相对的抵抗力，但对胶质霉素敏感。由于 PFA 烟雾是有毒的，因此在准备固定溶液时和固定步骤中始终使用烟气罩。处理 DAPI 时还要记得戴手套。

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

Detection of Mitochondria Membrane Potential to Study CLIC4 Knockdown-induced HN4 Cell Apoptosis In Vitro

Detection of Mitochondria Membrane Potential to Study CLIC4 Knockdown-induced HN4 Cell Apoptosis In Vitro

线粒体膜电位检测 CLIC4 击倒诱导 HN4 细胞凋亡的实验研究

Depletion of the mitochondrial membrane potential (MMP, &#916;&#936;m) is considered the earliest event in the apoptotic cascade. It even occurs ahead of ...

Immunophenotyping of Orthotopic Homograft (Syngeneic) of Murine Primary KPC Pancreatic Ductal Adenocarcinoma by Flow Cytometry

Immunophenotyping of Orthotopic Homograft Syngeneic of Murine Primary KPC Pancreatic Ductal Adenocarcinoma by Flow Cytometry

流式细胞术对小鼠原发性胰腺导管腺癌原位同种异体 (自体) 免疫分型的研究

Homograft (syngeneic) tumors are the workhorse of today&#39;s immuno-oncology (I/O) preclinical research. The tumor microenvironment (TME), particularly ...

Phospho Flow Cytometry with Fluorescent Cell Barcoding for Single Cell Signaling Analysis and Biomarker Discovery

磷流式细胞仪与荧光细胞条形码用于单细胞信号分析和生物标志物发现

Aberrant cell signaling plays a central role in cancer development and progression. Most novel targeted therapies are indeed directed at proteins and ...

Flow Cytometric Detection of Newly-formed Breast Cancer Stem Cell-like Cells After Apoptosis Reversal

细胞凋亡逆转后新形成乳腺癌干细胞样细胞的流式细胞仪检测

Cancer recurrence has long been studied by oncologists while the underlying mechanisms remain unclear. Recently, we and others found that a phenomenon ...

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

cd8+ t 细胞诱导体外肿瘤细胞凋亡的实时检测--研究肿瘤浸润髓内细胞的免疫抑制作用

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful ...

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 ...

Impedance-based Real-time Measurement of Cancer Cell Migration and Invasion

基于阻抗的癌细胞迁移和入侵的实时测量

Cancer arises due to uncontrolled proliferation of cells initiated by genetic instability, mutations, and environmental and other stress factors. These ...

In Vivo Immunogenicity Screening of Tumor-Derived Extracellular Vesicles by Flow Cytometry of Splenic T Cells

In Vivo Immunogenicity Screening of Tumor-Derived Extracellular Vesicles by Flow Cytometry of Splenic T Cells

体内 通过脾T细胞流式细胞术筛选肿瘤来源的细胞外囊泡的免疫原性

Immunogenic cell death of tumors, caused by chemotherapy or irradiation, can trigger tumor-specific T cell responses by releasing danger-associated ...

Simultaneous Imaging and Flow-Cytometry-based Detection of Multiple Fluorescent Senescence Markers in Therapy-Induced Senescent Cancer Cells

基于同步成像和流式细胞术检测治疗诱导的衰老癌细胞中的多种荧光衰老标志物

Chemotherapeutic drugs can induce irreparable DNA damage in cancer cells, leading to apoptosis or premature senescence. Unlike apoptotic cell death, ...