H2AX by Flow Cytometry
Following the induction of DNA double-stranded breaks (caused by genotoxins, UV or ionizing radiation exposure and replication errors) the histone protein H2AX becomes phosphorylated at serine 139 and rapidly accumulates at the site of DNA damage, forming distinct foci. This in turn facilitates the recruitment of DNA repair mechanisms and cell-cycle checkpoint factors.
As a result, the detection of H2AX foci has become a useful biomarker for DNA damage. High-content imaging and flow cytometry methods for H2AX phosphorylation are now widely used and such methods utilise immunofluorescent staining of H2AX (pS139) foci, to allow quantification of DNA damage. As H2AX phosphorylation is specific to DNA strand breaks, this assay provides a useful follow-up test to GreenScreen HC, BlueScreen HC and MNT to determine whether a positive result has a clastogenic mechanism.
- Sensitive assay for DNA damage
- Simple, robust and fast protocol
- Low compound requirement
- Provides mechanistic insight into genotoxicity positive results.
Cells are exposed to several concentrations of test chemical for 24 hours. Following removal of the test chemical, the cells are lysed and fixed. Cells are then incubated with a fluorescently labelled antibody which specifically binds to the phosphorylated H2AX foci. After the removal of the unbound antibody the cells are interrogated for the fluorescent label. An increase in fluorescence is directly proportional to an increase in H2AX phosphorylation. Positive and negative controls are included in each run of the assay as standard.
Data are collected using BD FACS CantoTM ll and BD FACSCaliburTM flow cytometers, with a minimum of 10,000 cellular events scored per dose. Exported data are then presented as H2AX fluorescence (y-axis) against test chemical concentration (x-axis). In addition, we are also able to show the H2AX expression as a proportion of the total DNA content. The study reports provide a detailed description of the study, data collection and results interpretation and are quality checked by a Gentronix expert flow cytometrist, independent to the study.