, 2002) Immunoblotting analysis: Here absolute amounts of γH2AX

, 2002). Immunoblotting analysis: Here absolute amounts of γH2AX protein are measured and compared to the total H2AX and H2A content. However, different cell types have different γH2AX/H2AX and H2AX/H2A ratios yielding as a result different absolute amounts of γH2AX for the same number of DSBs (Rogakou et al., 1998). Overall, microscopic analysis of γH2AX is considered to be more sensitive than other methods such as

flow cytometry (Kim et al., 2011). Initial microscopy developments in this area were limited BTK inhibitor in vivo to manual scoring of the samples which is restrictive in terms of sample generation (slide vs. microwell plate), operator time and subjectivity. New developments in the area of automated microscopy and image analysis software have increased the sensitivity of the results obtained by MAPK inhibitor HCS. Additionally, the use of microplates

and robotic systems has promoted the development of high throughput assays. Moreover, the use of software analysis allows objective quantitative scoring, avoiding operator subjectivity. The potential for multiplexing or evaluating various endpoints simultaneously is an attractive option as there would be a reduction in experimental time and resources. Therefore, from the current methods described above, HCS is considered a strong candidate for routine testing of γH2AX. In the last decade, the use of H2AX to assess DNA damage has grown exponentially as demonstrated by the number of publications (Fig. buy Decitabine 1A). This growth comes as a consequence of the diversification of scientific fields where H2AX is used (Fig. 1B). Initial studies were carried out in the field of radiation research, but once the relation between the phosphorylation of H2AX and DSBs was demonstrated

(Rogakou et al., 1998), the use of γH2AX soon expanded to other areas. The initial methodologies supported experimentation focused on DNA damage and repair mechanisms (Mukherjee et al., 2006, Marti et al., 2006, Celeste et al., 2003 and Bassing et al., 2003) to mention some. Other studies were orientated to assess the DNA damage potential of drugs, potency of chemotherapy agents and other medical materials (Tanaka et al., 2006, Ansteinsson et al., 2011 and Olive and Banath, 2009). Further optimisations in γH2AX detection allowed the use of this indicator of DSBs as a biomarker (Muslimovic et al., 2008 and Cornelissen et al., 2011). For example, Muslimovic et al. used non-fixed blood cells from irradiation patients to develop a biomarker that could potentially lead to modulation of radiological treatment (Muslimovic et al., 2008 and Johansson et al., 2011). The clinical use of γH2AX as a biomarker has been reviewed recently (Redon et al., 2010). In the field of genetic toxicology, Albino et al. proposed the use of γH2AX as a novel genotoxicity assay using flow cytometry (Albino et al., 2004) and was soon followed by Gallmeier et al. recommending immunocytochemistry (Gallmeier et al., 2005).

Leave a Reply

Your email address will not be published. Required fields are marked *

*

You may use these HTML tags and attributes: <a href="" title=""> <abbr title=""> <acronym title=""> <b> <blockquote cite=""> <cite> <code> <del datetime=""> <em> <i> <q cite=""> <strike> <strong>