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

Over a decade has passed since publication of the last review on ''Cytometry in cell necrobiology.'' During these years we have witnessed many substantial developments in the field of cell necrobiology such as remarkable advancements in cytometric technologies and improvements in analytical biochemistry. The latest innovative platforms such as laser scanning cytometry, multispectral imaging cytometry, spectroscopic cytometry, and microfluidic Lab-on-a-Chip solutions rapidly emerge as highly advantageous tools in cell necrobiology studies. Furthermore, we have recently gained substantial knowledge on alternative cell demise modes such as caspase-independent apoptosis-like programmed cell death (PCD), autophagy, necrosis-like PCD, or mitotic catastrophe, all with profound connotations to pathogenesis and treatment. Although detection of classical, caspase-dependent apoptosis is still the major ground for the advancement of cytometric techniques, there is an increasing demand for novel analytical tools to rapidly quantify noncanonical modes of cell death. This review highlights the key developments warranting a renaissance and evolution of cytometric techniques in the field of cell necrobiology. '

BH3 mimetics have been proposed as new anticancer therapeutics. They target anti-apoptotic Bcl-2 proteins, up-regulation of which has been implicated in the resistance of many cancer cells, particularly leukemia and lymphoma cells, to apoptosis. Using probabilistic computational modeling of the mitochondrial pathway of apoptosis, verified by single-cell experimental observations, we develop a model of Bcl-2 inhibition of apoptosis. Our results clarify how Bcl-2 imparts its anti-apoptotic role by increasing the time-to-death and cell-to-cell variability. We also show that although the commitment to death is highly impacted by differences in protein levels at the time of stimulation, inherent stochastic fluctuations in apoptotic signaling are sufficient to induce cell-to-cell variability and to allow single cells to escape death. This study suggests that intrinsic cell-to-cell stochastic variability in apoptotic signaling is sufficient to cause fractional killing of cancer cells after exposure to BH3 mimetics. This is an unanticipated facet of cancer chemoresistance.Electronic supplementary materialThe online version of this article (doi:10.1007/s10495-010-0515-7) contains supplementary material, which is available to authorized users.

Background: SYTO probes are gaining momentum as reliable and easy to use markers of apoptotic cell death, but the phenomenon underlying reduced SYTO fluorescence in apoptotic cells as compared with normal cells is still not fully elucidated. Herein, we attempt to provide further insights into mechanisms of reduced SYTO16 fluorescence during apoptosis. Methods: Human follicular lymphoma cell lines were subjected to diverse apoptotic and oncotic stimuli with subsequent multiparametric flow cytometric and fluorescence imaging analysis. SYTO green (SYTO11-16), TMRM, PI, 7AAD, and Hoechst 33342 probes were applied for multivariate analysis of temporal sequence of apoptotic events. Sorting of cells differing in the level of SYTO16 fluorescence and subsequent characterization of obtained subpopulations were also performed. Results: Loss of SYTO16 fluorescence (SYTO low /PI 1 events) has been observed in cells exposed to oncotic stimuli, whereas SYTO high /PI 1 events did not prevail at any treatment scenario. We tracked similarities and discrepancies

Peroxisome proliferator-activated receptor ␥ coactivator 1␣ (PGC-1␣) is an attractive candidate gene for type 2 diabetes, as genes of the oxidative phosphorylation (OXPHOS) pathway are coordinatively downregulated by reduced expression of PGC-1␣ in skeletal muscle and adipose tissue of patients with type 2 diabetes. Here we demonstrate that transgenic mice with activated polyamine catabolism due to overexpression of spermidine/spermine N 1 -acetyltransferase (SSAT) had reduced white adipose tissue (WAT) mass, high basal metabolic rate, improved glucose tolerance, high insulin sensitivity, and enhanced expression of the OXPHOS genes, coordinated by increased levels of PGC-1␣ and 5-AMP-activated protein kinase (AMPK) in WAT. As accelerated polyamine flux caused by SSAT overexpression depleted the ATP pool in adipocytes of SSAT mice and N 1 ,N 11 -diethylnorspermine-treated wild-type fetal fibroblasts, we propose that low ATP levels lead to the induction of AMPK, which in turn activates PGC-1␣ in WAT of SSAT mice. Our hypothesis is supported by the finding that the phenotype of SSAT mice was reversed when the accelerated polyamine flux was reduced by the inhibition of polyamine biosynthesis in WAT. The involvement of polyamine catabolism in the regulation of energy and glucose metabolism may offer a novel target for drug development for obesity and type 2 diabetes.Type 2 diabetes is a growing epidemic worldwide. Defects in insulin secretion and insulin action are fundamental disorders of this disease (30). Several mechanisms regulating insulin secretion and insulin action have been identified, but none of them is likely to explain completely the risk of type 2 diabetes. Previous studies have revealed novel mechanisms, distinct from the insulin signaling pathway, for type 2 diabetes. Mootha et al. (36) identified a set of genes involved in oxidative phosphorylation (OXPHOS), the expression of which was coordinately decreased in human diabetic muscle. Similarly, Patti et al. (40) found the downregulation of OXPHOS not only in individuals with type 2 diabetes but also in their first-degree relatives. In both of these studies, decreased peroxisome proliferator-activated receptor (PPAR) ␥ coactivator 1␣ (PGC-1␣) expression was responsible for the downregulation of OX PHOS genes. In addition, the expression of PGC-1␣ has been shown to be downregulated in white adipose tissue (WAT) of insulin-resistant (15) and morbidly obese (50) subjects.PGC-1␣ was first identified as a coactivator of PPAR␥ (45), and it plays a critical role in the regulation of adaptive thermogenesis. Subsequent studies have demonstrated that PGC-1␣ regulates mitochondrial biogenesis (49), uncoupling (45, 56), fatty acid oxidation (61), OXPHOS (36), glucose transport in muscle (35), hepatic gluconeogenesis (64), and skeletal muscle fiber-type switching (44). PGC-1␣ is highly expressed in brown adipose tissue (BAT), heart, and skeletal muscle and moderately expressed in liver, but a low expression level is found in WAT. The expression of PGC-1␣ is ind...