Increased lymphocyte death is a hallmark of human immunodeficiency virus (HIV) infection. Although virological factors have been linked to this phenomenon, increased cell death rates are still observed in treated individuals in which viral replication is halted. To understand the nature of this remaining altered cell death, we have developed a simple and fast assay to assess major cell death pathways in lymphocytes isolated from HIV-infected individuals. The combination of three factors: (i) antibody staining to identify CD3(+) CD4(+) and CD3(+) CD8(+) cells, (ii) assessment of mitochondrial and plasma membrane function using DiOC6(3) or JC-1 probes and vital dyes, and (iii) caspase inhibition, allowed for the quantification of caspase-independent and -dependent cell death in CD4 and CD8 T cells. The latter mechanism was divided in intrinsic and extrinsic apoptotic pathways according to the sensitivity of the dissipation of mitochondrial membrane potential to Z-VAD-fmk or Q-VD-oPH treatment. Our data show similar results for both caspase inhibitors in treated infected individuals, whereas Q-VD-oPH showed a more potent inhibition in viremic individuals, yielding lower levels of intrinsic apoptosis. Comparison of DiOC6(3) and JC-1 probes yielded similar results in CD4 T cells, allowing for a clear definition of death mechanism in these cells. However, in CD8 T-cells, JC-1 showed heterogeneous staining and detected significantly lower levels of cell death with a higher contribution of intrinsic apoptosis. In conclusion, we provide a simple method to assess CD4 T-cell death mechanisms in HIV-infected individuals. The reasons and consequences of mitochondrial heterogeneity in CD8 T-cells require further evaluation. © 2012 International Society for Advancement of Cytometry.

Fluorescence microscopy is commonly used for imaging live mammalian cells. Here, we describe studies aimed at revealing the potential genotoxic effects of standard fluorescence microscopy. To assess DNA damage, a high throughput platform for single cell gel electrophoresis is used (e.g., the CometChip). Light emitted by three standard filters was studied: (a) violet light [340-380 nm], used to excite DAPI and other blue fluorophores, (b) blue light [460-500 nm] commonly used to image green fluorescent protein (GFP) and Calcein AM, and (c) green light [528-553 nm], useful for imaging red fluorophores. Results show that exposure of samples to light during imaging is indeed genotoxic even when the selected wavelengths are outside the range known to induce significant damage levels. Shorter excitation wavelengths and longer irradiation times lead to higher levels of DNA damage. We have also measured DNA damage in cells expressing enhanced GFP or stained with Calcein AM, a widely used green fluorophore. Data show that Calcein AM leads to a synergistic increase in the levels of DNA damage and that even cells that are not being directly imaged sustain significant DNA damage from exposure to indirect light. The nature of light-induced DNA damage during imaging was assessed using the Fpg glycosylase, an enzyme that enables quantification of oxidative DNA damage. Oxidative damage was evident in cells exposed to violet light. Furthermore, the Fpg glycosylase revealed the presence of oxidative DNA damage in blue-light exposed cells for which DNA damage was not detected using standard analysis conditions. Taken together, the results of these studies call attention to the potential confounding effects of DNA damage induced by standard imaging conditions, and identify wavelength, exposure time, and fluorophore as parameters that can be modulated to reduce light-induced DNA damage. © 2013 International Society for Advancement of Cytometry.

On the cover: Wallace H. Coulter (1913-1998) was an iconic figure in the fields of cytometry and hematology. Over his lifetime, he initiated dozens of patents that transformed these fields from manual operations to automated systems. His entrepreneurial skills established Coulter Electronics and eventually Coulter Corporation into a billion dollar corporation. He spent his life working towards helping mankind by creating new diagnostic tools and research solutions. 2013 is the 100th anniversary of his birth. Painting of Wallace H. Coulter, courtesy of the Wallace H. Coulter Foundation. Cover design by Bärbel Beran. [www.beran-design.de].

Natural killer (NK) cells are capable of lysing their target cells with the help of perforin. The application of these cells for immunotherapy requires the estimation of their potency for the purpose of validation and batch-to-batch comparison. Cytotoxicity measurements have been carried out at only a few effector target ratios, therefore, allowing only semiquantitative assessment at best. By using a novel approach of varying the effector target ratio continuously and careful analysis of the experimental data after the reactions, we have achieved a precision necessary for constructing a mathematical model of cytotoxic reaction. Curve-fitting to experimental data indicates that NK cell cytotoxicity follows the law of mass action and fits the model of a single ligand-receptor interaction. The method allows to use the value of half-maximal lysis to describe the potency of cytotoxic NK cells numerically. © 2013 International Society for Advancement of Cytometry.

Only a few inventors can be said to have made as great an impact on mankind as Wallace Coulter. His inquisitive mind and ability to see well beyond what existed served him well for 40 years of inventing. So many of the fundamental tools that exist today in the area of hematology were derived from or driven by Coulter's inventions that he could be called the most technological innovator in the field of modern hematology. In achieving these discoveries Wallace Coulter was clearly capable of visualizing future opportunities that few others recognized. His vision was combined with an uncanny ability to translate his ideas into products. He developed a large number of tools that shaped the fields of cytometry, image analysis, and industrial materials. His understanding of the future power of computation drove him to link these technologies in a unique way. In the end, Coulter shaped the technologies that ultimately drove hematology in a new direction, one that remains on a critical pathway linking technology innovation all the way to true translational impact. It was said of Henry Ford that "[h]e has no notion that wealth has made him great, and any one who is imprest merely by his wealth bores him. In his personal contacts he likes to dodge the subject. He would prefer to talk with a machinist about machinery, or with somebody who likes birds about birds. In these contacts, he asks no deference; and if he gets it, he suspects it is mere deference to wealth, and that ends his interest."(1) The same could be said of Wallace Coulter, who, like Ford, understood the concepts of mass production and customer service. Coulter had the ability to recognize the opportunity and fulfill the need for development of a blood-cell counter that could be placed in every pathology laboratory, and in so doing transformed a field from a qualitative to a quantitative environment. Every person who has ever entered a medical lab, hospital, or doctor's office has felt the impact of Coulter's discovery. © 2013 International Society for Advancement of Cytometry.

For a little more than a century, fluorescence microscopy has been an essential source of major discoveries in cell biology. Recent developments improved both visualization and quantification by fluorescence microscopy imaging and established a methodology of fluorescence microscopy. By outlining basic principles and their historical development, I seek to provide insight into and understanding of the ever-growing tools of fluorescence microscopy. Thereby, this synopsis may help the interested researcher to choose a fluorescence microscopic method capable of addressing a specific scientific question. © 2013 International Society for Advancement of Cytometry.