Multiparameter detection of apoptosis using red-excitable SYTO probes

Functional assays allowing phenotypic characterization of different cell death parameters at a single-cell level are important tools for preclinical anticancer drug screening. Currently, the selection of cytometric assays is limited by the availability of fluorescent probes with overlapping spectral characteristics. Following on our earlier reports on green and orange fluorescent SYTO probes, we provide herein further insights into applicability of novel red-excitable SYTO stains (SYTO 17, 59-64) for multiparameter analysis of cell fate. In particular, SYTO 62 appears to be a spectrally favorable candidate. Using a correlative comparison between SYTO 16, Annexin V, YO-PRO 1, and fluorescently labeled inhibitors of caspases (FLICA), we demonstrate the specificity of SYTO 62 in detection of apoptotic cell death. Used in conjunction with FLICA or Annexin V, SYTO 62 stain proved amenable for multivariate kinetic analysis of apoptotic events. Considering simplicity of staining protocols, low cost, and avoidance of spectral compensation problems, we expect that red-excitable SYTO dyes will find a wide range of cytometric applications.