A cell undergoing apoptosis demonstrates multitude of characteristic morphological and biochemical features, which vary depending on the inducer of apoptosis, cell type and the “time window” at which the process of apoptosis is observed. Because the gross majority of apoptotic hallmarks can be revealed by flow and image cytometry, the cytometric methods become a technology of choice in diverse studies of cellular demise. Variety of cytometric methods designed to identify apoptotic cells, detect particular events of apoptosis and probe mechanisms associated with this mode of cell death have been developed during the past two decades. In the present review, we outline commonly used methods that are based on the assessment of mitochondrial transmembrane potential, activation of caspases, DNA fragmentation, and plasma membrane alterations. We also present novel developments in the field such as the use of cyanine SYTO and TO-PRO family of probes. Strategies of selecting the optimal multiparameter approaches, as well as potential difficulties in the experimental procedures, are thoroughly summarized.

In the past few years, overwhelming evidence has accrued that a high level of expression of the protein neuroglobin protects neurons in vitro, in animal models, and in humans, against cell death associated with hypoxic and amyloid insult. However, until now, the exact mechanism of neuroglobin’s protective action has not been determined. Using cell biology and biochemical approaches we demonstrate that neuroglobin inhibits the intrinsic pathway of apoptosis in vitro and intervenes in activation of pro-caspase 9 by interaction with cytochrome c. Using systems level information of the apoptotic signalling reactions we have developed a quantitative model of neuroglobin inhibition of apoptosis, which simulates neuroglobin blocking of apoptosome formation at a single cell level. Furthermore, this model allows us to explore the effect of neuroglobin in conditions not easily accessible to experimental study. We found that the protection of neurons by neuroglobin is very concentration sensitive. The impact of neuroglobin may arise from both its binding to cytochrome c and its subsequent redox reaction, although the binding alone is sufficient to block pro-caspase 9 activation. These data provides an explanation the action of neuroglobin in the protection of nerve cells from unwanted apoptosis.Electronic supplementary materialThe online version of this article (doi:10.1007/s10495-009-0436-5) contains supplementary material, which is available to authorized users.

An apoptosing cell demonstrates multitude of characteristic morphological and biochemical features, which vary depending on the stimuli and cell type. The gross majority of classical apoptotic hallmarks can be rapidly examined by flow and image cytometry. Cytometry thus became a technology of choice in diverse studies of cellular demise. A large variety of cytometric methods designed to identify apoptotic cells and probe mechanisms associated with this mode of cell demise have been developed during the past two decades. In the present chapter we outline a handful of commonly used methods that are based on the assessment of: mitochondrial transmembrane potential, activation of caspases, plasma membrane alterations and DNA fragmentation.

Apoptosis is a complex and finely controlled cell death process of great relevance in tissue homeostasis and pathogenesis. The majority of classical apoptotic features can be examined by flow as well as image cytometry. Therefore, cytometry has been used as a technology of choice in studies of tumor cell demise. As search for new and more effective anticancer agents is still ongoing, there is undoubtedly a need for further development of high-throughput screening platforms. Assays that allow multivariate characterization of cell death events in response to novel anticancer regimens are of particular significance. In this context, patented DNA-binding SYTO probes are gaining increasing interest as easy to use markers of caspase-dependent cell death. They are proving convenient for tracking apoptosis in diverse cell lines as well as in primary tumor samples. In this review, we outline most recent developments in the use of SYTO probes in cell necrobiology. We also present pilot characterization of novel SYTO orange stains (SYTO80 and SYTO81) and discuss their potential applications in cytometry of apoptosis. Finally, we provide a future outlook on SYTO probes in cytometric and microfluidics (Lab-on-a-Chip) high content analysis applications. ' 2008 International Society