评价以下电离辐射的空间分布的γH2AX
Summary
微观分析γH2AX灶，形成以下丝氨酸139磷酸化H2AX在DNA双链断裂，已成为辐射生物学中的一个非常宝贵的工具。在这里，我们使用一种抗体，单甲基化的组蛋白H3赖氨酸4作为一个积极抄写常染色质的表观遗传标记，以评估辐射诱导γH2AX形成的细胞核内的空间分布。
Abstract
早期的分子反应，DNA双链断裂（DSB的）磷酸化的SER – 139内的组蛋白H2AX 1,2终端SQEY图案的残留。 H2AX的磷酸化介导的磷脂，inosito 3 -激酶（PI3K）的蛋白质家族，共济失调毛细血管扩张症（ATM）的突变，DNA -蛋白激酶催化亚基，ATM和相关RAD3（ATR）的3。 H2AX的磷酸化形式，简称为γH2AX，蔓延到邻近地区的染色质的DSB网站，形成离散灶，很容易由3 immunofluorecence显微镜可视化。分析和定量γH2AX灶已被广泛用于评估DSB的形成和修复，特别是在电离辐射和评估各种修改化合物和细胞毒素化合物4辐射的功效， 。
鉴于这种双链断裂从头标志的精美的特异性和灵敏度，它在染色质中的DNA损伤和修复的过程提供新的见解。例如，在辐射生物学中心的范例是核DNA与放射敏感性的关键目标。事实上，在该领域的普遍共识在很大程度上被认为染色均匀的模板为DNA损伤与修复。然而，与γH2AX作为分子标记，DNA双链断裂，在γ射线照射，诱导γH2AX灶在常染色质和异染色质的形成悬殊的使用已被观察到5-7。最近，我们使用的抗体面板，无论是单，双或三甲基化组蛋白H3赖氨酸9（H3K9me1，H3K9me2，H3K9me3）后生印记构异染色质和转录沉默和赖氨酸4（H3K4me1，H3K4me2，H3K4me3 ），这是密切相关的积极抄写常染色质区域，调查γH2AX的空间分布， 电离辐射8。按照当时的想法，关于染色质生物学，我们的研究结果表明γH2AX的形成和活跃转录9之间的密切相关。在这里，我们展示了非贴壁细胞中γH2AX灶的检测和定量的免疫方法，特别注重与其他的表观遗传标记，图像分析和三维建模与合作本地化。
Protocol
细胞制备人红白血病K562细胞生长在10％（V / V）饱和湿度5％的CO 2环境中的胎牛血清和20毫克/毫升的庆大霉素的RPMI – 1640补充介质在37 ° C。 染色前约18小时，洗指数生长的细胞（5 × 10 5细胞/毫升是最优的）用磷酸盐缓冲液（PBS），悬浮在新媒体和返回到37 ° C，5％的CO 2。 两次用PBS离心洗涤细胞，在5分钟的转速约1500，并重新悬浮在新鲜的媒体。 …
Disclosures
The authors have nothing to disclose.
Acknowledgements
澳大利亚核科学与工程学院的支持，是公认的事实。 TCK的是AINSE奖项的收件人。表观医学实验室是支持由国家卫生和澳大利亚的医学研究理事会（566559）。这项工作是由“儿童权利公约”，生物医学成像发展有限公司，建立和下的澳大利亚政府S合作研究中心（CRC）计划的支持。 LM是由墨尔本研究所（墨尔本大学）和生物医学成像的华润补充奖学金支持。莫纳什显微成像（DRS斯蒂芬科迪ISKA卡迈克尔）的支持是这项工作的宝贵。
Materials
| Material Name | Type | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| Roswell Park Memorial Institute -1640 (RPMI-1640) | Growth medium | Invitrogen | 22400071 | RPMI-1640, pH 7.4 medium supplemented with 10% (v/v) fetal bovine, 2mM L-glutamine, 20μg/ml gentamicin, 20mM (HEPES) N-2-Hydroxyethylpiperazine-N’-2-Ethanesulfonic Acid |
| Fetal Bovine Serum (FBS) | Sigma-Aldrich | F2442 | ||
| Bovine Serum Albumin (BSA) | Sigma-Aldrich | A7906 | BSA (1%) is used to block any non-specific antibody binding. Primary and secondary antibodies are diluted in BSA. | |
| PBS (without Ca2+ and Mg2+) | Invitrogen | 17-517Q | ||
| Trypan blue | Sigma Aldrich | T6146 | Used to distinguish between live and dead cells. | |
| Triton X-100 | Reagent | Sigma-Aldrich | T8787 | Triton X-100 (0.1%) used to permeabilise cells. |
| Paraformaldehyde | Reagent | Sigma-Aldrich | 158127 | Paraformaldehyde (4%) used to fix cells. |
| Mouse monoclonal anti-phospho histone-H2AX antibody | Primary Antibody | Millipore, USA | 16193 | Dilution of primary antibody (1:500), in 1% BSA. |
| HistoneH3 (Mono-methyl K4) | Primary Antibody | Abcam, Cambridge, UK | AB8895 | |
| Alexa Fluor 488 goat anti-mouse IgG (H+L) | Secondary Antibody | Invitrogen, USA | 11029 | Dilution of secondary antibody (1:500), in 1% BSA. |
| Alexa Fluor 546 goat anti-rabbit IgG (H+L) | Secondary Antibody | Invitrogen, USA | 11035 | |
| TOPRO3 | DNA Stain | Invitrogen, USA | T3605 | TOPRO3 is a DNA dye with an Abs/Em of 642/661 nm. DAPI could be used if the confocal microscope is equipped with a 405 nm laser. |
| ProLong Gold | Anti-fade solution | Invitrogen | P36930 | This glycerol based mounting medium must be used with an oil based lense that matches its refractive index. |
| Polylysine slides | Menzel-Gläser, Germany | |||
| Coverslips (22x50mm) | Coverslips | Menzel-Gläser, Germany | CS2250100 | |
| Tissue Culture Flask, Vented Cap | Culture Flask | BD Falcon | 353112 | |
| Shandon Cytospin 4 | ThermoElectron Corp | |||
| Cytofunnels | Shandon, Inc. | |||
| Filter Cards | Shandon, Inc | 353025 | ||
| Coplin Jar, glass | Grale Scientific P/L | 1771-OG | ||
| Staining Trough | Grale Scientific P/L | V1991.99 | ||
| PAP Pen | Zymed Laboratories, Invitrogen | 008877 | ||
| Gammacell 1000 Elite Irradiator | Gamma Irradiator | Nordion International Inc. | ||
| Zeiss LSM 510 Meta Confocal | Confocal Microscope | Equipped with 3 lasers: 488 nm, 543 nm and 633 nm. | ||
| Metamorph | Software for Imaging analysis | Molecular Devices, USA |
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Tags
Spatial DistributionγH2AXIonizing RadiationDNA Double-strand Breaks (DSBs)Ser-139 ResidueHistone H2AXPhosphorylationPhosphatidyl-inosito 3-kinase (PI3K) FamilyAtaxia Telangiectasia Mutated (ATM)DNA-protein Kinase Catalytic SubunitATM And RAD3-related (ATR)Immunofluorescence MicroscopyγH2AX FociDSB Formation And RepairRadiation Modifying CompoundsCytotoxic CompoundsDNA Damage And RepairChromatin Templateγ-irradiation-induced γH2AX Foci
Cite This Article
Vasireddy, R. S., Tang, M. M., Mah, L., Georgiadis, G. T., El-Osta, A., Karagiannis, T. C. Evaluation of the Spatial Distribution of γH2AX following Ionizing Radiation. J. Vis. Exp. (42), e2203, doi:10.3791/2203 (2010).