title: Multispectral imaging flow cytometry reveals distinct frequencies of γ-H2AX foci induction in DNA double strand break repair defective human cell lines.
abstract: The measurement of γ-H2AX foci induction in cells provides a sensitive and reliable method for the quantitation of DNA damage responses in a variety of cell types. Accurate and rapid methods to conduct such observations are desirable. In this study, we have employed the novel technique of multispectral imaging flow cytometry to compare the induction and repair of γ-H2AX foci in three human cell types with different capacities for the repair of DNA double strand breaks (DSB). A repair normal fibroblast cell line MRC5-SV1, a DSB repair defective ataxia telangiectasia (AT5BIVA) cell line, and a DNA-PKcs deficient cell line XP14BRneo17 were exposed to 2 Gy gamma radiation from a (60)Cobalt source. Thirty minutes following exposure, we observed a dramatic induction of foci in the nuclei of these cells. After 24 hrs, there was a predictable reduction on the number of foci in the MRC5-SV1 cells, consistent with the repair of DNA DSB. In the AT5BIVA cells, persistence of the foci over a 24-hr period was due to the failure in the repair of DNA DSB. However, in the DNA-PKcs defective cells (XP14BRneo17), we observed an intermediate retention of foci in the nuclei indicative of partial repair of DNA DSB. In summary, the application of imaging flow cytometry has permitted an evaluation of foci in a large number of cells (20,000) for each cell line at each time point. This provides a novel method to determine differences in repair kinetics between different cell types. We propose that imaging flow cytometry provides an alternative platform for accurate automated high through-put analysis of foci induction in a variety of cell types.
authors.affiliation: Brunel Institute of Cancer Genetics and Pharmacogenomics, Division of Biosciences, School of Health Sciences and Social Care, Brunel University, Uxbridge, Middlesex UB8 3PH, United Kingdom.
authors.forename: Emma C
authors.initials: EC
authors.lastname: Bourton
authors.forename: Piers N
authors.initials: PN
authors.lastname: Plowman
authors.forename: Sheba Adam
authors.initials: SA
authors.lastname: Zahir
authors.forename: Gonul Ulus
authors.initials: GU
authors.lastname: Senguloglu
authors.forename: Hiba
authors.initials: H
authors.lastname: Serrai
authors.forename: Graham
authors.initials: G
authors.lastname: Bottley
authors.forename: Christopher N
authors.initials: CN
authors.lastname: Parris
mesh_terms: D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism / Q000528:radiation effects; D053903:DNA Breaks, Double-Stranded / Q000528:radiation effects; D004260:DNA Repair / Q000528:radiation effects; D004307:Dose-Response Relationship, Radiation; D005434:Flow Cytometry / Q000379:methods*; D005453:Fluorescence; D005720:Gamma Rays; D006657:Histones / Q000378:metabolism*; D006801:Humans; D019044:Image Cytometry / Q000379:methods*
chemical_list: C468783:H2AX protein, human; D006657:Histones