Towards an understanding of apoptosis detection by SYTO dyes

Wlodkowic, Donald; Skommer, Joanna; Pelkonen, Jukka

doi:10.1002/cyto.a.20366

Cited by 41 publications

(111 citation statements)

References 31 publications

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“…In virtually all reports, apoptotic cells are stained using a method that includes cell washing, and results are expressed as percentage of all cells analyzed (13). Our comparison shows dramatic differences between the wash and the no-wash procedures ( Figs.…”

Section: Wash and No-wash Cell Preparation To Analyze Apoptosis In Thmentioning

confidence: 95%

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Novel single‐platform multiparameter FCM analysis of apoptosis: Significant differences between wash and no‐wash procedure

et al. 2010

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FCM is a generally accepted tool to analyze apoptosis. Unfortunately, the cell preparation of all commercial kits available includes cell washing known to cause cell loss which is most likely to affect apoptotic cells in particular. To address this, we developed a seven-color single-platform no-wash analysis technique and compared the results with those from an analogous procedure including cell washing. A five-color mAb cocktail was employed to address target cells by surface labeling, Yo-PRO-1 1 and DAPI were used to discriminate apoptotic and necrotic from viable cells. Cells were quantified on the basis of internal-standard fluorescent beads. Jurkat cells ACC 282 treated with camptothecin were employed to establish the staining procedure, which was then applied to blood cells collected by extracorporeal apheresis and treated with UV irradiation. Data evaluation showed that although each method by itself was highly reproducible (R 2 5 0.973), the numbers of apoptotic cells detected with the no-wash procedure were significantly higher than those obtained after cell washing (P 5 6.6 E 25 , Wilcoxon Test). In addition, the observed differences increased with higher cell numbers (Bland and Altmann). We conclude that the described test is a feasible and reliable tool for apoptosis measurement and it provides results that are definitely closer to the truth than those obtained from kits that require cell washing. ' 2010 International Society for Advancement of CytometryKey terms multicolor flow cytometry; apoptosis; Trucount TM ; single platform; Yo-PRO-1 1 APOPTOSIS plays a major role in various physiological and pathological processes.Cell demise has been investigated in different fields, also including extracorporeal photopheresis (ECP), a therapeutic intervention used in several auto immune disorders, diseases with pathogenic T cell involvement, and chronic graft-versus-host disease (GvHD) (1). Collected cells are reinfused after in vitro exposure to 8-methoxypsoralen and ultraviolet A (UVA) light. One known effect of ECP on PBMC is the induction of apoptosis (2,3), which can be analyzed by FCM, a generally accepted and reliable tool for detection of apoptosis on single-cell level (4,5). Several assays for FCM analysis of different stages of programmed cell death are commercially available but all of them include at least one centrifugation step. Hypothesizing that, after apoptosis-inducing treatment, part of the PBMC will be disrupted by centrifugation and thus will not be present for FCM analysis, our intention was to establish a single-platform, no-wash, and multicolor FCM methodology to quantify apoptotic T cells in ECP products.To discriminate viable from early and late apoptotic/necrotic cells, we tested several markers. We favored the YO-PRO-1 1 /DAPI combination which allows discrimination of apoptotic from necrotic cells. YO-PRO-1, a DNA-intercalating dye excited at 488 nm, is a reliable marker for FCM analysis of apoptosis (6,7). DAPI, a nuclear dye excited at 405 nm, which is commonly used in ...

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Section: Wash and No-wash Cell Preparation To Analyze Apoptosis In Thmentioning

confidence: 95%

“…In contrast to our approach, however, they applied cell washing steps at various time points during cell culture or preparation (6,16,17) or rinsed cells with PBS after induction of apoptosis (13). Our intention was to demonstrate that in particular apoptotic cells are lost by cell washing.…”

Section: Technical Notementioning

confidence: 98%

Novel single‐platform multiparameter FCM analysis of apoptosis: Significant differences between wash and no‐wash procedure

et al. 2010

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“…Another approach has been the use of SYTO dyes, originally thought of as nucleic acid binding dyes that could be used to detect apoptosis (23)(24)(25)(26). SYTO dyes have been used as a cheaper replacement for annexin V (7,26). Interestingly, SYTO16 was shown not to significantly reduce during sodium azide or heat shock induction of oncosis, the cells rapidly lost membrane integrity, a classic profile of oncosis (7).…”

mentioning

confidence: 99%

“…SYTO dyes have been used as a cheaper replacement for annexin V (7,26). Interestingly, SYTO16 was shown not to significantly reduce during sodium azide or heat shock induction of oncosis, the cells rapidly lost membrane integrity, a classic profile of oncosis (7). A few earlier studies have tried to find a means to show a difference between cells undergoing apoptosis and oncosis without much success by standard flow cytometry (12,13,22).…”

mentioning

confidence: 99%

“…Measurement of mitochondrial function was reported more recently to show differences in cells undergoing apoptosis and oncosis in a drug dose response study after 24 h exposure (13). Although most earlier studies concluded that it is not possible to distinguish between cells generated from an apoptotic or oncotic process the recent use of SYTO16 does afford a means to show differences in cells undergoing apoptosis and oncosis by standard flow cytometry in a time dependent manner (7,12,22). …”

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Real‐time flow cytometry for the kinetic analysis of oncosis

Warnes

Martins

2011

Cytometry Pt A

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The standard method of distinguishing apoptotic and oncotic cells has been by microscopic analysis of nuclei and cell membrane morphology. Thus a rapid test for analyzing large numbers of cells in the study of cell necrobiology has not been possible until the recent advent of the Amnis Image-stream and real-time Lab-on-a-Chip technologies. An interesting difference between apoptosis and oncosis is that they are ATP dependent and independent processes, respectively. Here we describe an assay measuring real-time kinetic changes in the potential differences of the inner mitochondrial membrane (mmp) and the plasma membrane (pmp) in cells immediately before and after the addition of the inducing agent. Live cells were loaded with carbocyanine dye DiIC 1 (5) and bis-oxonol (DiBAC 4 (5)) to measure mmp and pmp in conjunction with annexin V-FITC and DAPI labeling for gating out annexin V binding cells and dead cells respectively. Live cells gave specific membrane signatures in response to apoptotic or oncotic reagents in real-time. Apoptosis showed little change in mmp and pmp signals over the course of 25 min, the mitochondria only showed a slight hyperpolarization. In contrast chemical treatment with oxidative phosphorylation blocker, sodium azide (SA) caused an immediate hyperpolarization spike followed by a complete abrogation of mmp over a 25 min time course. Treatment with SA (1%) also caused plasma membrane depolarization. Likewise detergent (0.01% Triton X-100) treatments also caused abrogation of mmp and depolarization of pmp. Whereas heat shock (428C) treatment showed only a slight mitochondrial membrane potential depolarization. These flow cytometric observations were confirmed by confocal microscopy. This novel real-time kinetic assay measuring mitochondrial and plasma membrane potential changes has important implications in the field of cell necrobiology in that it allows the researcher to differentiate apoptotic and oncotic processes in an immediate manner for the first time. '

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SYTO Probes: Markers of Apoptotic Cell Demise

Wlodkowic

Skommer

2007

CP Cytometry

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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.

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Towards an understanding of apoptosis detection by SYTO dyes

Cited by 41 publications

References 31 publications

Novel single‐platform multiparameter FCM analysis of apoptosis: Significant differences between wash and no‐wash procedure

Novel single‐platform multiparameter FCM analysis of apoptosis: Significant differences between wash and no‐wash procedure

Real‐time flow cytometry for the kinetic analysis of oncosis

SYTO Probes: Markers of Apoptotic Cell Demise

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