电离辐射的反应定量的γH2AX灶
Summary
DNA双链分子标记γH2AX形成条纹的量化已成为辐射生物学中的一个非常宝贵的工具。在这里，我们表现出免疫荧光法检测细胞对辐射的暴露后γH2AX灶的定量使用。
Abstract
DNA双链断裂（DSB的），这是无论是内源性代谢过程或外源性诱导，是最关键的DNA损伤一个方面的生存和保持基因组完整性。早期反应的双链断裂感应是组蛋白H2A变体，H2AX的磷酸化，丝氨酸139残留，在高度保守的C -末端SQEY图案，形成γH2AX<sup> 1</sup>。 （ATR）的双链断裂感应后，H2AX的迅速磷酸化的磷脂，inosito 3 – 激酶（PIKK）蛋白家族，共济失调毛细血管扩张症（ATM）的突变，DNA -蛋白激酶催化亚基和ATM和RAD3相关<sup> 2</sup>。通常情况下，只有少数几个碱基对（BP）在DSB有牵连，但是，有显着的信号放大，信号和修复DNA损伤的染色质修饰的重要性。 H2AX的磷酸化介导主要由ATM染色邻近地区的利差，影响约0.03％的细胞总H2AX的每DSB<sup> 2,3</sup>。这相当于约2000 H2AX的分子跨越〜2 MBP染色质的地区，周围的DSB网站和离散γH2AX灶形成的结果，可以很容易地可视化和免疫荧光显微镜定量磷酸化<sup> 2</sup>。 γH2AX在DSB的损失反映了修复，但是，有一些争论什么定义完整的双链断裂的修复，它已经提出，重新加入DNA的两条链是足够的，然而，它也被建议重新恢复压实原有的染色质状态是必要的<sup> 4-8</sup>。 γH2AXdisappearence涉及至少部分，去磷酸化磷酸酶，磷酸酶2A和磷酸4C<sup> 5,6</sup>。此外，去除再分配涉及与H2A.Z组蛋白交流的γH2AX有牵连<sup> 7,8</sup>。重要的是，γH2AX灶的定量分析，导致了广泛的应用在医学和核研究。在这里，我们展示了初始γ辐照壁人角质形成细胞DNA的损伤γH2AX灶的检测和定量的评价最常用的免疫方法<sup> 9</sup>。
Protocol
Discussion
暴露于电离辐射（γ射线），γH2AX灶形式迅速和疫源地号码后达到最大，在30-60 分钟之间2。因此，1小时后照射时间点反映初始DSB的形成。我们的实验中，我们已经用2 Gy的临床相关的辐射剂量。然而，该方法可用于辐射剂量高达4戈瑞检测初始DSB的形成;灶明显的重叠排除在较高剂量准确定量。可用于更高的辐射剂量辐照后的潜伏期较长时间，γH2AX灶因维修，造成量化的数字。通常情况下，4…
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）。 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 |
|---|---|---|---|---|
| Keratinocyte-Serum Free Medium (K-SFM) | Media | Invitrogen | 17005042 | Keratinocyte media supplemented with human recombinant Epidermal Growth Factor 1-53 (EGF 1-53), and Bovine Pituitary Extract (BPE). |
| Lab Tek lI Chamber Slides (8-well) | Chamber Slides | Nunc Denmark | NUN154534 | |
| Coverslips (22x50mm) | Coverslips | Menzel-Gläser | CS2250100 | |
| 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 | ||
| 0.5% Trypsin-EDTA x10 | Invitrogen | 15400-054 | Trypsin-EDTA (0.05%) used to detach cells from culture flasks. | |
| 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 | 16193 | Dilution of primary antibody (1:500), in 1% BSA. |
| Alexa Fluor 488 goat anti-mouse IgG (H+L) | Secondary Antibody | Invitrogen | 11029 | Dilution of secondary antibody (1:500), in 1% BSA. |
| TOPRO3 | DNA Stain | Invitrogen | T3605 | DNA stain commonly used: 4,6-diamidino-2-phenylindole dihydrochloride (DAPI). Can only be used with microscopes with the appropriate excitation laser. |
| ProLong Gold | Anti-fade solution | Invitrogen | P36930 | Refractive index of 1.42 at 20°C. |
| Tissue Culture Flask, Vented Cap | Culture Flask | BD Falcon | 353112 | |
| Tissue Culture Dish (150x25mm) | Petridish | BD Falcon | 353025 | |
| Coplin Jar, glass | Grale Scientific P/L | 1771-OG | ||
| Staining Trough | Grale Scientific P/L | V1991.99 | ||
| Gammacell 1000 Elite Irradiator | Gamma Irradiator | Nordion International Inc. | ||
| Zeiss LSM 510 Meta Confocal | Confocal Microscope | |||
| Metamorph | Software for Imaging analysis | Molecular Devices, USA |
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