An Automated Microscopic Scoring Method for the &amp;#947;-H2AX Foci Assay in Human Peripheral Blood Lymphocytes

Nair, Shankari; Cairncross, Samantha; Miles, Xanthene; Engelbrecht, Monique; Plessis, Peter du; Bolcaen, Julie; Fisher, Randall; Ndimba, Roya; Cunningham, Charnay; Martínez‐López, Wilner; Schunck, Christian H.; Vandevoorde, Charlot

doi:10.3791/62623

Cited by 6 publications

(6 citation statements)

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“…In vivo , p53 mRNA, p53 protein, γ-H2AX protein and lncRNA TUG1 expression also increased after subarachnoid anesthesia with 0.5% bup treatment, which was consistent with the in vitro experiment. We presume that γ-H2AX is expressed during the late phase in the chain reaction of double-stranded DNA repair [ 60 , 61 ] . We believe that the p53 protein activates and upregulates the expression of TUG1 and promotes the repair of damaged DNA, thereby reducing DNA damage and the overall level of cell damage.…”

Section: Discussionmentioning

confidence: 99%

P53 and taurine upregulated gene 1 promotes the repair of the DeoxyriboNucleic Acid damage induced by bupivacaine in murine primary sensory neurons

et al. 2022

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The research aimed to explore the biological role of p53 protein and long non-coding RNA (lncRNA) taurine upregulated gene 1 (TUG1) in bupivacaine (bup)-induced neurotoxicity. Our work treated dorsal root ganglion (DRG) cells with bup, detected cell viability through CCK-8, apoptosis through TUNEL assays, DeoxyriboNucleic Acid (DNA) damage through γ-H2AX protein and comet assay, including p53 mRNA, protein and TUG1 expression through q-PCR and western blot, furthermore, cell viability and DNA damage were determined after the silencing of p53 and TUG1, biological information and TUG1 FISH combined with p53 protein immunofluorescence (IF) was performed to determine the cellular localization of these molecule. In vivo experiments, we explored the impact of intrathecal injection of bup on p53 mRNA and protein, TUG1, γ-H2AX protein expression. The results showed that bup was available to signally decreased cell viability, promoted apoptosis rate and DNA damage, additionally, bup increased p53 mRNA and protein and TUG1 expression. P53 siRNA and TUG1 siRNA significantly increased DNA damage. Furthermore, bioinformatics analysis and colocalization experiments revealed that the p53 protein is a transcription factor of TUG1, in vivo experiment, intrathecal injection of bup increased the p53 mRNA, p53 protein, TUG1 and γ-H2AX protein in the murine DRG. In this study, it was found p53 and TUG1 promote the repair of the DNA damage induced by bup in murine dorsal root ganglion cells, suggesting a new strategy for the amelioration of bup-induced neurotoxicity.

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Section: Discussionmentioning

confidence: 99%

P53 and taurine upregulated gene 1 promotes the repair of the DeoxyriboNucleic Acid damage induced by bupivacaine in murine primary sensory neurons

et al. 2022

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“…Spectacular advances have been achieved in recent years in terms of sensitivity and interface after uniform staining using cytogenetic slides or flow cytometers (DAPI) [ 21 , 73 , 74 , 75 ]. However, this automation concerns only the scoring of micronuclei and does not make it possible to differentiate the origin of micronuclei because centromere staining has not yet been implemented.…”

Section: Automation Of Biological Dosimetry Methodsmentioning

confidence: 99%

“…Direct molecular consequences such as single and double-strand breaks in the DNA molecule detected by fluorochrome-labeled antibodies specific for, e.g., γH2AX, MR11 proteins, or p53-binding protein (53BP1) that are markers of double-strand breaks (DSB) [ 19 , 20 ]. The preparation and analysis of the immunostainings can be automated [ 4 , 21 ]. The proteins are useful markers of DSB ( Table 1 ) in analyses of putative biological effects of low doses of irradiation [ 22 ] and for the triage of exposed populations [ 23 , 24 ].…”

Section: Cytogenetic Markers Of Irradiationmentioning

confidence: 99%

High Resolution and Automatable Cytogenetic Biodosimetry Using In Situ Telomere and Centromere Hybridization for the Accurate Detection of DNA Damage: An Overview

M’Kacher

Colicchio

Junker

et al. 2023

IJMS

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In the event of a radiological or nuclear accident, or when physical dosimetry is not available, the scoring of radiation-induced chromosomal aberrations in lymphocytes constitutes an essential tool for the estimation of the absorbed dose of the exposed individual and for effective triage. Cytogenetic biodosimetry employs different cytogenetic assays including the scoring of dicentrics, micronuclei, and translocations as well as analyses of induced premature chromosome condensation to define the frequency of chromosome aberrations. However, inherent challenges using these techniques include the considerable time span from sampling to result, the sensitivity and specificity of the various techniques, and the requirement of highly skilled personnel. Thus, techniques that obviate these challenges are needed. The introduction of telomere and centromere (TC) staining have successfully met these challenges and, in addition, greatly improved the efficiency of cytogenetic biodosimetry through the development of automated approaches, thus reducing the need for specialized personnel. Here, we review the role of the various cytogenetic dosimeters and their recent improvements in the management of populations exposed to genotoxic agents such as ionizing radiation. Finally, we discuss the emerging potentials to exploit these techniques in a wider spectrum of medical and biological applications, e.g., in cancer biology to identify prognostic biomarkers for the optimal triage and treatment of patients.

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“…The microscope-based method for the gamma-H2AX assay is also amenable to automated image analysis ( 38 , 39 ) . In this case, the number of foci can be detected on slides after fluorescent labelling with anti-gamma-H2AX antibodies ( 20 , 40 ) .…”

Section: Slide-based Analysismentioning

confidence: 99%

Development of high-throughput systems for biodosimetry

Wilkins,

Beaton-Green

2023

Radiation Protection Dosimetry

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Biomarkers for ionising radiation exposure have great utility in scenarios where there has been a potential exposure and physical dosimetry is missing or in dispute, such as for occupational and accidental exposures. Biomarkers that respond as a function of dose are particularly useful as biodosemeters to determine the dose of radiation to which an individual has been exposed. These dose measurements can also be used in medical scenarios to track doses from medical exposures and even have the potential to identify an individual’s response to radiation exposure that could help tailor treatments. The measurement of biomarkers of exposure in medicine and for accidents, where a larger number of samples would be required, is limited by the throughput of analysis (i.e. the number of samples that could be processed and analysed), particularly for microscope-based methods, which tend to be labour-intensive. Rapid analysis in an emergency scenario, such as a large-scale accident, would provide dose estimates to medical practitioners, allowing timely administration of the appropriate medical countermeasures to help mitigate the effects of radiation exposure. In order to improve sample throughput for biomarker analysis, much effort has been devoted to automating the process from sample preparation through automated image analysis. This paper will focus mainly on biological endpoints traditionally analysed by microscopy, specifically dicentric chromosomes, micronuclei and gamma-H2AX. These endpoints provide examples where sample throughput has been improved through automated image acquisition, analysis of images acquired by microscopy, as well as methods that have been developed for analysis using imaging flow cytometry.

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An Automated Microscopic Scoring Method for the γ-H2AX Foci Assay in Human Peripheral Blood Lymphocytes

Cited by 6 publications

References 0 publications

P53 and taurine upregulated gene 1 promotes the repair of the DeoxyriboNucleic Acid damage induced by bupivacaine in murine primary sensory neurons

P53 and taurine upregulated gene 1 promotes the repair of the DeoxyriboNucleic Acid damage induced by bupivacaine in murine primary sensory neurons

High Resolution and Automatable Cytogenetic Biodosimetry Using In Situ Telomere and Centromere Hybridization for the Accurate Detection of DNA Damage: An Overview

Development of high-throughput systems for biodosimetry

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