Highly Sensitive Automated Method for DNA Damage Assessment : Gamma-H2AX Foci Counting and Cell Cycle Sorting
Hernández Garcia, Laia (Universitat Autònoma de Barcelona. Departament de Biologia Cel·lular, de Fisiologia i d'Immunologia)
Terradas, Mariona (Universitat Autònoma de Barcelona. Departament de Biologia Cel·lular, de Fisiologia i d'Immunologia)
Martín Flix, Marta (Universitat Autònoma de Barcelona. Departament de Biologia Cel·lular, de Fisiologia i d'Immunologia)
Tusell Padrós, Laura (Universitat Autònoma de Barcelona. Departament de Biologia Cel·lular, de Fisiologia i d'Immunologia)
Genescà, Anna (Anna) (Universitat Autònoma de Barcelona. Departament de Biologia Cel·lular, de Fisiologia i d'Immunologia)

Date:	2013
Abstract:	Phosphorylation of the H2AX protein is an early step in the double strand break (DSB) repair pathway; therefore, phosphorylated histone (γH2AX) foci scoring is widely used as a measure for DSBs. Foci scoring is performed either manually or semi-automatically using hand-operated capturing and image analysis software. In general, both techniques are laborious and prone to artifacts associated with manual scoring. While a few fully automated methods have been described in the literature, none of them have been used to quantify γH2AX foci in combination with a cell cycle phase analysis. Adding this feature to a rapid automated γH2AX foci quantification method would reduce the scoring uncertainty that arises from the variations in the background level of the γH2AX signal throughout the cell cycle. The method was set up to measure DNA damage induced in human mammary epithelial cells by irradiation under a mammogram device. We adapted a FISH (fluorescent in situ hybridization) Spot-counting system, which has a slide loader with automatic scanning and cell capture system throughout the thickness of each cell (z-stack), to meet our assay requirements. While scanning the sample, the System classifies the selected nuclei according to the signal patterns previously described by the user. For our purposes, a double staining immunofluorescence was carried out with antibodies to detect γH2AX and pericentrin, an integral component of the centrosome. We could thus distinguish both the number of γH2AX foci per cell and the cell cycle phase. Furthermore, restrictive settings of the program classifier reduced the "touching nuclei" problem described in other image analysis software. The automated scoring was faster than and as sensitive as its manually performed counterpart. This system is a reliable tool for γH2AX radio-induced foci counting and provides essential information about the cell cycle stage. It thus offers a more complete and rapid assessment of DNA damage.
Rights:	Aquest document està subjecte a una llicència d'ús Creative Commons. Es permet la reproducció total o parcial, la distribució, la comunicació pública de l'obra i la creació d'obres derivades, fins i tot amb finalitats comercials, sempre i quan es reconegui l'autoria de l'obra original.
Language:	Anglès
Document:	Article ; recerca ; Versió publicada
Subject:	Gamma-H2AX ; Cell cycle ; Automated screening method ; Dosimetry ; DNA damage ; Mammography
Published in:	International journal of molecular sciences, Vol. 14 Núm. 8 (july 2013) , p. 15810-15826, ISSN 1422-0067

DOI: 10.3390/ijms140815810
PMID: 23903043

17 p, 1.8 MB

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