Fast Detection of DNA Damage

Darzynkiewicz, Zbigniew, Zhao, Hong, Dorota Halicka, H., Pozarowski, Piotr, Lee, Brian, Zbigniew Darzynkiewicz, Hong Zhao, H. Dorota Halicka, Piotr Pozarowski, Brian Lee

Activation of caspases is a characteristic event of apoptosis. Various cytometric methods distinguishing this event have been developed to serve as specific apoptotic markers for the assessment of apoptotic frequency within different cell populations. The method described in this chapter utilizes fluorochrome labeled inhibitors of caspases (FLICA) and is applicable to fluorescence microscopy, flow- and imaging-cytometry as well as to confocal imaging. Cell-permeant FLICA reagents tagged with carboxyfluorescein or sulforhodamine, when applied to live cells in vitro or in vivo, exclusively label the cells that are undergoing apoptosis. The FLICA labeling methodology is rapid, simple, robust, and can be combined with other markers of cell death for multiplexed analysis. Examples are presented on FLICA use in combination with a vital stain (propidium iodide), detection of the loss of mitochondrial electrochemical potential, and exposure of phosphatidylserine on the outer surface of plasma cell membrane using Annexin V fluorochrome conjugates. FLICA staining followed by cell fixation and stoichiometric staining of cellular DNA demonstrate that FLICA binding can be correlated with the concurrent analysis of DNA ploidy, cell cycle phase, DNA fragmentation, and other apoptotic events whose detection requires cell permeabilization. The "time window" for the detection of apoptosis with FLICA is wider compared to the Annexin V binding, making FLICA a preferable marker for the detection of early phase apoptosis and therefore more accurate for quantification of apoptotic cells. Unlike many other biomarkers of apoptotic cells, FLICAs can be used to detect apoptosis ex vivo and in vivo in different tissues.