Synchronization of cells and nuclei is a powerful technique for the exact study of regulatory mechanisms and for understanding cell cycle events. Counterflow centrifugal elutriation is a biophysical cell separation technique in which cell size and sedimentation density differences of living cells are exploited to isolate subpopulations in various stages of cell cycle. Here, a protocol is described for the separation of phase-enriched subpopulations from exponentially growing Chinese hamster ovary cells at high-resolution power of elutriation. The efficiency of elutriation is confirmed by measuring the DNA content fluorimetrically and by flow cytometry. The resolution power of elutriation is demonstrated by the ability to fractionate nuclei of murine pre-B cells. The installation and elutriation by collecting 16-30 synchronized fractions, including particle size analysis, can be achieved in 4-5 h.

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Sixteen groups of mice were fed diets containing different selenium species to compare their toxicity. Inorganic sodium selenate and sodium hydroselenite, elementary nanoSe, organic Sel-Plex, and Lacto-MicroSelenium were administered for 14 d at concentrations of 0.5, 5, and 50 ppm Se, equivalent to 0.5, 5, and 50 mg Se/kg food, corresponding to an estimated 4, 40, and 400 µg/kg body weight/d Se uptake, respectively. At the end of the treatment, body, liver, spleen, kidney, heart, and brain weights were measured, mice were subjected to necropsy, and histological examinations were performed on the liver. At lower Se doses (0.5 and 5 ppm) a moderate reduction was observed in the number of bone marrow and white blood cells and in granulocyte-macrophage colony-forming units (GM-CFUs) relative to the untreated control group of mice. A comparison of lowest toxic doses of sodium selenite in mice (0.5 ppm) and mallard (10 ppm) indicates that birds are more resistant to Se than rodents. In mice, a small but measurable weight loss was observed after 5 ppm selenate and LactoMicroSe treatment. The most significant changes took place after 50-ppm administration in body and spleen weight, hematology, and liver histology. Toxicity was more pronounced when inorganic Se was applied than after subacute application of Sel-Plex, nanoSe, or LactoMicroSe. To summarize the effects, the authors' 14-d murine subacute toxicity study showed that the toxicity of Se species decreased in the following order: selenate > selenite > nanoSe > Sel-Plex > LactoMicroSe.

In concert with the increased understanding that there are many ways for cells to die, several methods have been developed to detect cell death. The classification of cell death posed some difficulties that were overcome by implementing strict selection criteria that should also apply to the detection methods. The selection of assays is based on morphological criteria and distinguishable marks of apoptotic patways. The detection of apoptosis includes methods related to membrane alterations, DNA fragmentation, cytotoxicity and cell proliferation, mitochondrial damage, immunological detection and mechanism based assays. Other less frequently used detections of apoptosis are: (a) light-scattering flow cytometry to avoid underestimating the extent and timing of apoptosis, (b) time-lapse microscopy perfusion platform to support the temporal aspects of detection, to measure cell surface area and cellular adhesion, and (c) genotoxicity specific chromatin changes. Attention is called to the advantages and limitations of various methods.

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