The data suggest that the post-irradiation time used for estimation of radiosensitivity at therapeutically relevant low doses (e.g., <3 Gy) in proliferating cells by scoring residual foci should be limited by the duration of the cell cycle, and that direct comparison of the kinetics of DSB repair and disappearance of DSB-co-localizing foci is not possible. Therefore, results obtained from the counting of foci should be interpreted with caution in terms of DSB repair.

Heat shock (HS) is one of the best-studied exogenous cellular stresses. The cellular response to HS utilizes ancient molecular networks that are based primarily on the action of stress-induced heat shock proteins and HS factors. However, in one way or another, all cellular compartments and metabolic processes are involved in such a response. In this review, we aimed to summarize the experimental data concerning all aspects of the HS response in mammalian cells, such as HS-induced structural and functional alterations of cell membranes, the cytoskeleton and cellular organelles; the associated pathways that result in different modes of cell death and cell cycle arrest; and the effects of HS on transcription, splicing, translation, DNA repair, and replication.

Ionizing radiation induced foci (IRIF) are considered the most sensitive indicator for DNA double-strand break (DSB) detection. Monitoring DSB induction by low doses of ionizing radiation is important due to the increasing exposure in the general population. cH2AX and 53BP1 are commonly used molecular markers for in situ IRIF assessment. Imaging flow cytometry (IFC) via ImageStream system provides a new opportunity in this field. We analyzed the formation of 53BP1, cH2AX foci and their co-localization induced by c-rays (2, 5, 10, 50, 200 cGy) in human lymphocytes using ImageStream and the automated microscopic system Metafer. We observed very similar sensitivity of both systems for the detection of endogenous and low-dose-induced IRIF. Statistically significant induction of cH2AX foci was found at doses of 2 and 10 cGy using ImageStream and Metafer, respectively. Statistically significant induction of 53BP1 foci was evident at doses !5 cGy when analyzed by IFC. Analysis of the colocalizing foci by ImageStream and Metafer showed statistical significance at doses !2 cGy, suggesting that foci co-localization is a sensitive parameter for DSB quantification. Assessment of cH2AX, 53BP1 foci and their co-localization by Metafer and ImageStream showed similar linear dose responses in the low-dose range up to 10 cGy, although IFC showed slightly better resolution for IRIF in this dose range. At higher doses, IFC underestimated IRIF numbers. Using the imaging ability of ImageStream, we introduced an optimized assay by gating cH2AX foci positive (with 1 or more cH2AX foci) and negative (cells without foci) cells. This assay resulted in statistically significant IRIF induction at doses ! 5cGy and a linear dose response up to 50 cGy. In conclusion, we provide evidence for the use of IFC as an accurate high throughput assay for the prompt detection and enumeration of endogenous and low-dose induced IRIF. V C 2015 International Society for Advancement of Cytometry Key terms imaging flow cytometry; ImageStream; Metafer; human lymphocytes; cH2AX; 53BP1; ionizing radiation DNA double strand breaks (DSB) are known as the most deleterious type of DNA damage induced by ionizing radiation and other genotoxic agents. Misrepaired or unrepaired DSB lead to genome rearrangements and contribute to carcinogenesis. Currently, the most sensitive and specific method for DSB analysis is enumeration of small repair centers formed at the locations of DSB. These centers are known as ionizing radiation induced foci (IRIF) or DNA repair foci. IRIF are dynamic structures containing thousands of copies of proteins involved in various aspects of DSB repair and DNA damage response. Phosphorylated histone H2AX variant (cH2AX) and p53 binding protein 1 (53BP1) are commonly accepted molecular markers of IRIF (1,2). Upon DSB induction, histone H2AX molecules become phosphorylated within