SYTO Probes: Markers of Apoptotic Cell Demise

Abstract

As mechanistic studies on tumor cell death advance towards their ultimate translational goal, there is a need for specific, rapid, and high‐throughput analytical tools to detect diverse cell demise modes. Patented DNA‐binding SYTO probes, for example, are gaining increasing interest as easy‐to‐use markers of caspase‐dependent apoptotic cell death. They are proving convenient for tracking apoptosis in diverse hematopoietic cell lines and primary tumor samples, and, due to their spectral characteristics, appear to be useful for the development of multiparameter flow cytometry assays. Herein, several protocols for multiparametric assessment of apoptotic events using SYTO probes are provided. There are protocols describing the use of green fluorescent SYTO 16 and red fluorescent SYTO 17 dyes in combination with plasma membrane permeability markers. Another protocol highlights the multiparametric use of SYTO 16 dye in conjunction with the mitochondrial membrane potential sensitive probe, tetramethylrhodamine methyl ester (TMRM), and the plasma membrane permeability marker, 7‐aminoactinomycin D (7‐AAD). Curr. Protocol. Cytom. 42:7.33.1‐7.33.12. © 2007 by John Wiley & Sons, Inc.

Apoptosis and Beyond: Cytometry in Studies of Programmed Cell Death

“…Exploitation of SYTO probes to cytometric detection of apoptosis started in 1990s (Frey, 1995; Poot et al , 1997) and is slowly gaining appreciation as an easy to perform, live-cell assay (Poot et al , 1997; Schuurhuis et al , 2001). Although, the fundamental mechanism underlying differential staining of SYTO-labeled apoptotic versus viable cells still remains uncertain, several hypotheses have been raised in the recent years (reviewed in Wlodkowic and Skommer, 2007). …”

“…The latter population is characterized by initial loss of SYTO 11 fluorescence and preservation of plasma membrane integrity (SYTO dim /PI neg events; A). Cells in later stages of apoptosis feature progressive loss of SYTO fluorescence and gain bright PI staining (SYTO neg /PI + events; N) (Wlodkowic and Skommer, 2007; Wlodkowic et al , 2007b). Of note, the primary necrotic cells will also fall into the last group with minimal SYTO 11 and bright PI fluorescence.…”

“…Moreover, one should always bear in mind that results obtained using SYTO-based assays may vary when compared to assays detecting different cellular processes. Results acquired with SYTO probes should, therefore, never be considered conclusive without verification by independent methods (Wlodkowic and Skommer, 2007; Wlodkowic et al , 2007b). …”

Dynamic analysis of apoptosis using cyanine SYTO probes: From classical to microfluidic cytometry

“…Staining with SYTOX green (30 nM), SYTOX red (10 nM) or YO-PRO 1 (250 nM) was performed as previously described [6,7]. …”

Median Effect Dose and Combination Index Analysis of Cytotoxic Drugs Using Flow Cytometry

“…The SYTO probes bind DNA with low affinity in live or dead cells (Eray et al, 2001;Poot et al, 1997). They are combined with high affinity cell-impermeant dyes to discriminate live from dead cells (Wlodkowic & Skommer, 2007). For example cell-permeant SYTO-red will stain live and dead cells with red fluorescence binding with low affinity to DNA, but if used together with SYTOX-green dead cells will be green fluorescent, because SYTOX-green has much higher affinity for DNA and will displace the low affinity SYTOred.…”