Adapting the γ-H2AX assay for automated processing in human lymphocytes. 1. Technological aspects.
Adapting the γ-H2AX assay for automated processing in human lymphocytes. 1. Technological aspects.
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DOI：
10.1667/rr2125.1
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发表时间：
2011-03
影响因子：
3.4
通讯作者：
Garty G
中科院分区：
文献类型：
--
作者：
Turner HC;Brenner DJ;Chen Y;Bertucci A;Zhang J;Wang H;Lyulko OV;Xu Y;Shuryak I;Schaefer J;Simaan N;Randers-Pehrson G;Yao YL;Amundson SA;Garty G
免疫荧光法检测γ-H_2AX是一种可靠、灵敏的定量检测辐照样品中DNA双链断裂的方法。由于H_2AX的磷酸化与辐射剂量高度线性，这一公认的生物标志物目前被用于辐射生物剂量测定。在高通量微创辐射生物剂量测定中心，我们开发了一种全自动高通量系统，RABIT(快速自动化生物剂量测定工具)，可用于从指棒提取的血液样本中测量γ-H2AX的产量。RABIT工作站旨在完全自动化γ-H2AX免疫细胞化学程序，从在涂有肝素的聚氯乙烯毛细管中分离人血淋巴细胞，到免疫标记γ-H2AX蛋白和图像采集以确定荧光产量。高产量是通过使用专门制造的机器人、在96孔过滤器底板中处理淋巴细胞和高速成像实现的。本研究的目的是优化和验证RABIT系统的性能，以便以多孔格式重复性和定量地检测淋巴细胞中的γ-H_2AX总荧光。我们的生物剂量学平台的验证是通过线性检测外周血样本中γ-H2AX荧光随剂量的增加而实现的，该外周血样本在0至8Gy射线范围内受到γ射线照射。这项研究首次证明了我们基于机器人的生物剂量测量工作站的优化和使用，成功地定量了照射后外周淋巴细胞中的γ-H_2AX总荧光。
The immunofluorescence-based detection of γ-H2AX is a reliable and sensitive method for quantitatively measuring DNA double-strand breaks (DSBs) in irradiated samples. Since H2AX phosphorylation is highly linear with radiation dose, this well-established biomarker is in current use in radiation biodosimetry. At the Center for High-Throughput Minimally Invasive Radiation Biodosimetry, we have developed a fully automated high-throughput system, the RABIT (Rapid Automated Biodosimetry Tool), that can be used to measure γ-H2AX yields from fingerstick-derived samples of blood. The RABIT workstation has been designed to fully automate the γ-H2AX immunocytochemical protocol, from the isolation of human blood lymphocytes in heparin-coated PVC capillaries to the immunolabeling of γ-H2AX protein and image acquisition to determine fluorescence yield. High throughput is achieved through the use of purpose-built robotics, lymphocyte handling in 96-well filter-bottomed plates, and high-speed imaging. The goal of the present study was to optimize and validate the performance of the RABIT system for the reproducible and quantitative detection of γ-H2AX total fluorescence in lymphocytes in a multiwell format. Validation of our biodosimetry platform was achieved by the linear detection of a dose-dependent increase in γ-H2AX fluorescence in peripheral blood samples irradiated ex vivo with γ rays over the range 0 to 8 Gy. This study demonstrates for the first time the optimization and use of our robotically based biodosimetry workstation to successfully quantify γ-H2AX total fluorescence in irradiated peripheral lymphocytes.