Dataset Information

Development of a high-throughput ?-H2AX assay based on imaging flow cytometry.

ABSTRACT:

Background

Measurement of ?-H2AX foci levels in cells provides a sensitive and reliable method for quantitation of the radiation-induced DNA damage response. The objective of the present study was to develop a rapid, high-throughput ?-H2AX assay based on imaging flow cytometry (IFC) using the ImageStream®^X Mk II (ISX) platform to evaluate DNA double strand break (DSB) repair kinetics in human peripheral blood cells after exposure to ionizing irradiation.

Methods

The ?-H2AX protocol was developed and optimized for small volumes (100??L) of human blood in Matrix™ 96-tube format. Blood cell lymphocytes were identified and captured by ISX INSPIRE™ software and analyzed by Data Exploration and Analysis Software.

Results

Dose- and time-dependent ?-H2AX levels corresponding to radiation exposure were measured at various time points over 24?h using the IFC system. ?-H2AX fluorescence intensity at 1?h after exposure, increased linearly with increasing radiation dose (R² =?0.98) for the four human donors tested, whereas the dose response for the mean number of ?-H2AX foci/cell was not as robust (R² =?0.81). Radiation-induced ?-H2AX levels rapidly increased within 30?min and reached a maximum by ~?1?h, after which time there was fast decline by 6?h, followed by a much slower rate of disappearance up to 24?h. A mathematical approach for quantifying DNA repair kinetics using the rate of ?-H2AX decay (decay constant, K_dec), and yield of residual unrepaired breaks (F_res) demonstrated differences in individual repair capacity between the healthy donors.

Conclusions

The results indicate that the IFC-based ?-H2AX protocol may provide a practical and high-throughput platform for measurements of individual global DNA DSB repair capacity which can facilitate precision medicine by predicting individual radiosensitivity and risk of developing adverse effects related to radiotherapy treatment.

SUBMITTER: Lee Y

PROVIDER: S-EPMC6704696 | biostudies-literature | 2019 Aug

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Development of a high-throughput γ-H2AX assay based on imaging flow cytometry.

Lee Younghyun Y Wang Qi Q Shuryak Igor I Brenner David J DJ Turner Helen C HC

Radiation oncology (London, England) 20190822 1

<h4>Background</h4>Measurement of γ-H2AX foci levels in cells provides a sensitive and reliable method for quantitation of the radiation-induced DNA damage response. The objective of the present study was to develop a rapid, high-throughput γ-H2AX assay based on imaging flow cytometry (IFC) using the ImageStream®<sup>X</sup> Mk II (ISX) platform to evaluate DNA double strand break (DSB) repair kinetics in human peripheral blood cells after exposure to ionizing irradiation.<h4>Methods</h4>The γ-H ...[more]

PMID: 31438980

Similar Datasets

Project description:Mitochondria are highly dynamic organelles whose fusion and fission play an increasingly important role in a number of both normal and pathological cellular functions. Despite the increased interest in mitochondrial dynamics, robust, and quantitative methods to analyze mitochondrial fusion and fission activity in intact cells have not been developed. The current state-of-the art method to measure mitochondrial fusion activity is the polyethylene glycol (PEG) fusion assay in which cells expressing distinct mitochondrially-targeted fluorescent proteins (FPs) are fused together and mitochondrial fusion activity is determined by the rate at which color mixing occurs. Although this assay is useful, cell-cell fusion events are rare, and finding the number of fused cells required to generate statistically rigorous data is both tedious and time-consuming. Furthermore, the data-collection methods available for fluorescence microscopy lead to inherent selection biases that are difficult to control for. To that end, we have developed an unbiased and high-throughput method to detect, image, and analyze fused cells using the Amnis ImagestreamX™ MKII. With IDEAS™ software, we developed algorithms for identifying the fused cells (two nuclei within a single cell), distinguishing them from cell aggregates. Additionally, using the fluorescence localization of the mitochondrially-targeted fluorescent proteins (YFP and DsRed), we applied a modified co-localization algorithm to identify those cells that had a high co-localization score indicating mitochondrial fusion activity. These algorithms were tested using negative controls (FPs associated with fusion deficient mitochondria) and positive controls (cells expressing both FPs in the same mitochondria). Once validated these algorithms could be applied to test samples to evaluate the degree of mitochondrial fusion in cells with various genetic mutations. Ultimately, this new method is the first robust, high-throughput way to directly measure mitochondrial fusion in intact cells. Given how many cellular processes are being linked mitochondrial dynamics, this technique will provide a powerful new tool in the study of this important organelle. © 2016 International Society for Advancement of Cytometry.

Project description:The androgen receptor (AR) is a steroid hormone receptor which regulates transcription of androgen-sensitive genes and is responsible for the development and maintenance of male secondary sexual characteristics. Chemicals that interfere with AR activity may lead to pathological conditions in androgen-sensitive tissues. A variety of reporter systems have been developed, driven by androgen-sensitive promoters, which screen for chemicals that modulate androgenic activity. We have developed a flexible, high-throughput AR transcriptional activation assay, designated the Multifunctional Androgen Receptor Screening (MARS) assay, to facilitate the identification of novel modulators of AR transcriptional activity using flow cytometry. Androgen-independent human prostate cancer-derived PC3 cells were transiently cotransfected with an expression vector for the wild-type human AR and an androgen-sensitive promoter regulating the expression of destabilized enhanced GFP (dsEGFP). The transfected cells were stimulated with established androgenic and antiandrogenic compounds and assessed for increased or decreased dsEGFP expression. To screen for antagonists of AR transcription, the AR agonist R1881 was coadministered at submaximal concentrations with potential AR antagonists. The assay was formatted for high-throughput screening using the HyperCyt flow cytometry system. Agents with established androgenic and antiandrogenic activity were used for validation of the MARS assay. AR agonists were found to potently induce dsEGFP. Furthermore, AR agonists induced dsEGFP expression in a dose-dependent manner. Alternatively, AR antagonists blocked dsEGFP expression when coadministered with low-dose R1881, which also occurred in a dose-dependent manner. Modulators of AR transcriptional activity can be successfully identified by the MARS assay, utilizing a rapid, flexible, sensitive, and high-throughput format. Dose-response curves can be successfully generated for these compounds, allowing for an assessment of potency. Because of its simplicity and high-throughput compatibility, the MARS assay and HyperCyt system combined with flow cytometric analysis represents a valuable and novel addition to the current repertoire of AR transcriptional activation screening assays.