Adapting the γ-H2AX Assay for Automated Processing in Human Lymphocytes. 1. Technological Aspects
- 1 March 2011
- journal article
- Published by Radiation Research Society in Radiation Research
- Vol. 175 (3), 282-290
- https://doi.org/10.1667/rr2125.1
Abstract
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.Keywords
This publication has 37 references indexed in Scilit:
- THE RABIT: A RAPID AUTOMATED BIODOSIMETRY TOOL FOR RADIOLOGICAL TRIAGEHealth Physics, 2010
- THE LYMPHOCYTE CYTOKINESIS-BLOCK MICRONUCLEUS CYTOME ASSAY AND ITS APPLICATION IN RADIATION BIODOSIMETRYHealth Physics, 2010
- Development of a robotically-based automated biodosimetry tool for high-throughput radiological triageInternational Journal of Biomechatronics and Biomedical Robotics, 2010
- Late Residual γ-H2AX Foci In Murine Skin are Dose Responsive and Predict RadiosensitivityIn VivoRadiation Research, 2010
- H2AX: functional roles and potential applicationsChromosoma, 2009
- γ-H2AX as a biomarker of DNA damage induced by ionizing radiation in human peripheral blood lymphocytes and artificial skinAdvances in Space Research, 2009
- γH2AX and cancerNature Reviews Cancer, 2008
- An optimized method for detecting gamma-H2AX in blood cells reveals a significant interindividual variation in the gamma-H2AX response among humansNucleic Acids Research, 2007
- Pathways of DNA Double-Strand Break Repair during the Mammalian Cell CycleMolecular and Cellular Biology, 2003
- Involvement of H2AX in the DNA Damage and Repair ResponseCancer Biology & Therapy, 2003