title: A modified fluorimetric neutral filter elution method for analyzing radiation-induced double strand break and repair.
abstract: Neutral filter elution assay is one of the methods used for detection of DNA double strand breaks (DSBs). However, it is laborious, expensive, and hazardous (radiolabeled precursors for DSB detection and scintillation counter for quantification), making it a less preferred method for DSB detection. In the present study, an attempt was made to improve the existing neutral filter elution assay by making use of fluorescent dye (PicoGreen) and microfiltration assembly for eluting the fragmented DNA, thereby reducing the cost and time required for the assay. We studied the effect of dye dilution, pH conditions, and cell number as a part of method standardization. X-ray dose-response and repair kinetics in lymphocytes as well as cell lines were studied for validating the sensitivity of the assay. A linear dose-response relationship for DSBs was observed at a cell number of 4×10(5)cells, a dye dilution of 500-fold, and at pH 10. Repair kinetics revealed a time-dependent repair of DSBs up to 360 min of posttreatment, indicating its usefulness in DSB repair studies. In conclusion, the present modified method is more efficient (in terms of cell number), cost effective, less time-consuming, and less hazardous compared to the existing method.
authors: Division of Radiobiology & Toxicology, Manipal Life Sciences Centre, Manipal University, Manipal, India.
authors: Venkatesh H
authors: VH
authors: Goutham
authors: Mumbrekar D
authors: MD
authors: Kamalesh
authors: Parashiva K
authors: PK
authors: Guruprasad
authors: Manjunath B
authors: MB
authors: Vadhiraja
authors: Kapaettu
authors: K
authors: Satyamoorthy
authors: Sadashiva
authors: S
authors: Rao Bola Satish
mesh_terms: D045744:Cell Line, Tumor; D004247:DNA / Q000737:chemistry / Q000378:metabolism / Q000528:radiation effects*; D053903:DNA Breaks, Double-Stranded; D004260:DNA Repair; D005374:Filtration / Q000379:methods; D005456:Fluorescent Dyes / Q000737:chemistry; D005470:Fluorometry / Q000379:methods*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009930:Organic Chemicals / Q000737:chemistry
chemical_list: D005456:Fluorescent Dyes; D009930:Organic Chemicals; C099476:PicoGreen; D004247:DNA