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| Format: | Artículo científico |
| Langue: | en |
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Academia Brasileira de Ciências
2016
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| Accès en ligne: | https://www.redalyc.org/articulo.oa?id=32744812011 |
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- Radiation dose determines the method for quantification of DNA double strand breaks TANJA BULAT OTILIJA KETA LELA KORIĆANAC JELENA ŽAKULA IVAN PETROVIĆ ALEKSANDRA RISTIĆ-FIRA DANIJELA TODOROVIĆ Multidisciplinaria (Ciencias Naturales y Exactas) γ H2AX Western blot ApoTome software AxioImagerA1 microscope immunofluorescence microscopy Ionizing radiation induces DNA double strand breaks (DSBs) that trigger phosphorylation of the histone protein H2AX ( γ H2AX). Immuno fluorescent staining visualizes formation of γ H2AX foci, allowing their quanti fi cation. This method, as opposed to Western blot assay and Flow cytometry, provides more accurate analysis, by showing exact position and intensity of fluorescent signal in each single cell. In practice there are problems in quantification of γ H2AX. This paper is based on two issues: the determination of which technique should be applied concerning the radiation dose, and how to analyze fluorescent microscopy images obtained by different microscopes. HTB140 melanoma cells were exposed to γ -rays, in the dose range from 1 to 16 Gy. Radiation effects on the DNA level were analyzed at different time intervals after irradiation by Western blot analysis and immuno fluorescence microscopy. Immunochemically stained cells were visualized with two types of microscopes: AxioVision (Zeiss, Germany) microscope, comprising an ApoTome software, and AxioImagerA1 microscope (Zeiss, Germany). Obtained results show that the level of γ H2AX is time and dose dependent. Immuno fluorescence microscopy provided better detection of DSBs for lower irradiation doses, while Western blot analysis was more reliable for higher irradiation doses. AxioVision microscope containing ApoTome software was more suitable for the detection of γ H2AX foci. 2016 artículo científico 0001-3765 https://www.redalyc.org/articulo.oa?id=32744812011 en http://www.redalyc.org/revista.oa?id=327 Anais da Academia Brasileira de Ciências application/pdf Academia Brasileira de Ciências Anais da Academia Brasileira de Ciências (Brasil) Num.1 Vol.88