Annexins are a family of calcium-dependent phospholipid-binding proteins that preferentially bind phosphatidylserine (PS).
Under normal physiologic conditions, PS is predominantly located in the inner leaflet of the plasma membrane. Upon initiation of apoptosis, PS loses its asymmetric distribution across the phospholipid bilayer and is translocated to the extracellular membrane leaflet, marking cells as targets of phagocytosis. The translocation of PS is utilised as a biomarker for apoptosis through the PS binding characteristics of Annexin proteins. Recombinant Annexin-V protein, which has been fluorescently labelled, binds to the externalised PS of apoptotic cells; labelling these cells with a fluorescent signal that allows their detection by flow cytometry.
Cells are exposed to test chemical for a number of time points in the presence of growth medium. Typically this will include time points at 16 and 24 hours, with additional time points included as required. Following the exposure period, cells are harvested by centrifugation, washed and then exposed to Annexin V (conjugated to a fluorochrome). Following the removal of unbound antibody, cells can be exposed to a vital stain prior to flow cytometric assessment. A vital stain is used to separate early apoptotic cells that have externalised PS but are not permeable (Annexin V positive), from necrotic and late-stage apoptotic cells that are permeable and therefore allow Annexin V to bind internal as well as external PS (Annexin V and vital stain positive). An increase in fluorescence is proportional to the amount of Annexin V bound to the cell. Positive and vehicle controls are included in the assay as standard.
ResultsData are collected using either a FACS Canto II or a FACS Calibur flow cytometer with a minimum of 10,000 cellular events collected per test chemical dose, at each time point. Exported data are then presented as Annexin V fluorescence (y-axis) against test chemical concentration (x-axis). Additionally, bivariate data are also displayed for Annexin V and vital stain fluorescence. The study report provides a detailed description of the study, data collection and results interpretation and is quality checked by a Gentronix expert flow cytometrist, independent to the study.