title: Development of an automated gamma-H2AX immunocytochemistry assay.
abstract: gamma-H2AX is emerging as an important marker of ionizing radiation-induced double-strand breaks. Development of a significantly automated method to quantify gamma-H2AX would have broad application in assessing physiological responses to radiation exposure. PC-3 and DU145 prostate cancer cells grown on glass cover slips and 96-well plates were irradiated and assessed for gamma-H2AX focus formation by immunofluorescence analysis. The gamma-H2AX immunofluorescence staining was performed either manually or by using a preprogrammed automated robotic liquid handling system. A computer-controlled charge-coupled device camera acquired images serially throughout the thickness of each cell. Image analysis was performed manually and/or with automated image segmentation software. A robust relationship between radiation dose and gamma-H2AX focus numbers was demonstrated with both manual and automated image analysis methods, with excellent agreement observed between the two techniques. The r(2) correlation coefficients and Z factors exceeded 0.9 and 0.5, respectively, when gamma-H2AX focus formation was correlated with radiation dose using the automated technique. Inhibition of gamma-H2AX foci by drugs readily detected with this assay. Robotic specimen preparation with automated image acquisition and analysis can be used to quantify gamma-H2AX foci in irradiated cells, and the results agree well those obtained by manual counts. These data suggest that this assay has an excellent signal-to-noise ratio and is suitable for high-throughput applications.
authors.affiliation: Department of Radiation Oncology, Mount Sinai School of Medicine, New York, New York, USA.
authors.forename: Yen-Nien
authors.initials: YN
authors.lastname: Hou
authors.forename: Amir
authors.initials: A
authors.lastname: Lavaf
authors.forename: Delphine
authors.initials: D
authors.lastname: Huang
authors.forename: Sheila
authors.initials: S
authors.lastname: Peters
authors.forename: Rumana
authors.initials: R
authors.lastname: Huq
authors.forename: Victor
authors.initials: V
authors.lastname: Friedrich
authors.forename: Barry S
authors.initials: BS
authors.lastname: Rosenstein
authors.forename: Johnny
authors.initials: J
authors.lastname: Kao
mesh_terms: D001331:Automation; D015415:Biomarkers / Q000378:metabolism; D045744:Cell Line, Tumor; D053903:DNA Breaks, Double-Stranded / Q000528:radiation effects; D005240:Feasibility Studies; D006657:Histones / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods*; D008853:Microscopy; D010766:Phosphorylation; D011829:Radiation Dosage
chemical_list: D015415:Biomarkers; C468783:H2AX protein, human; D006657:Histones