The assay can detect carcinogens that act directly on the DNA (clastogens) ( Kirpnick et al., 2005). The methodology has been modified to support microwell plate use thereby increasing throughput ( Hafer et al., 2010). However, there are still concerns about the cell wall permeability of the yeast and the perceived relevance of the cell system ( Lynch et al., 2011). There are 2 major limitations to the current in vitro mammalian genotoxicity assays: Low throughput: this is mainly linked to the manual scoring that limits

large scale screening in terms of time. In the last few years, some technologies have been developed to increase the throughput. For example, automated flow cytometric analysis is used to score in vitro micronucleus samples ( Bryce et al., 2007). This methodology could potentially be used as a pre-screening tool while awaiting further validation, as detailed in a recent review ( Avlasevich check details et al., 2011). The γH2AX assay could be of potential use in overcoming GDC 0068 the 2 major limitations mentioned above. There are several methods for detecting γH2AX and these have evolved to become simpler, quicker and more automated. Initially, γH2AX detection employed acetic acid-urea-triton and acid-urea-cetyltrimethylammonium bromide polyacrylamide gel electrophoresis (aut-aucPAGE), a two-dimensional gel analysis to detect the level of phosphorylated H2AX. Gels from

untreated mammalian cell cultures were compared to gels generated using radiated cultures. The gels from the treated cells showed an additional shadowed area identified as a region containing the γH2AX protein which migrates through the gel differently than non-phosphorylated H2AX (Rogakou et al., 1998). However, after the initial development of this approach, immunocytochemical detection as described by Rogakou et al. became the primary method of detection, as it is several orders of magnitude more sensitive and has the potential for quantitation (Rogakou et al., 1999), (Sedelnikova et al., 2002). This method is based on the use of a γH2AX-specific monoclonal fluorophore-coupled

antibody. Once γH2AX presence has been detected by the Ketotifen antibody based technique, the results can be quantified using various methods. These approaches have been discussed extensively in a previous review (Bonner et al., 2008) and are summarised briefly below: Immunofluorescence analysis: a phosphospecific antibody is used to detect γH2AX, the antibody does not bind to any non-phosphorylated H2AX. This antibody can either be directly labelled with a fluorophore reporter or detected by addition of a secondary, fluorophore-labelled antibody. The stained γH2AX can then be analysed by manual or automated scoring. – Manual scoring: the stained cells are evaluated by eye using a fluorescence microscope. This method will only be able to give qualitative results, i.e. presence or absence of fluorescence. Additionally, the number of foci per cell could be counted.