Reactivation of the cell cycle, including DNA replication, might play a major role in Alzheimer's disease (AD). A more than diploid DNA content in differentiated neurons might alternatively result from chromosome mis-segregation during mitosis in neuronal progenitor cells. It was our objective to distinguish between these two mechanisms for aneuploidy and to provide evidence for a functional cell cycle in AD. Using slide-based cytometry, chromogenic in situ hybridization, and PCR amplification of alu-repeats, we quantified the DNA amount of identified cortical neurons in normal human brain and AD and analyzed the link between a tetraploid DNA content and expression of the early mitotic marker cyclin B1. In the normal brain, the number of neurons with a more than diploid content amounts to ϳ10%. Less than 1% of neurons contains a tetraploid DNA content. These neurons do not express cyclin B1, most likely representing constitutional tetraploidy. This population of cyclin B1-negative tetraploid neurons, at a reduced number, is also present in AD. In addition, a population of cyclin B1-positive tetraploid neurons of ϳ2% of all neurons was observed in AD. Our results indicate that at least two different mechanisms need to be distinguished giving rise to a tetraploid DNA content in the adult brain. Constitutional aneuploidy in differentiated neurons might be more frequent than previously thought. It is, however, not elevated in AD. In addition, in AD some neurons have re-entered the cell cycle and entirely passed through a functional interphase with a complete DNA replication.

Microfabricated flow cytometers can detect, count, and analyze cells or particles using microfluidics and electronics to give impedance-based characterization. Such systems are being developed to provide simple, low-cost, label-free, and portable solutions for cell analysis. Recent work using microfabricated systems has demonstrated the capability to analyze micro-organisms, erythrocytes, leukocytes, and animal and human cell lines. Multifrequency impedance measurements can give multiparametric, high-content data that can be used to distinguish cell types. New combinations of microfluidic sample handling design and microscale flow phenomena have been used to focus and position cells within the channel for improved sensitivity. Robust designs will enable focusing at high flowrates while reducing requirements for control over multiple sample and sheath flows. Although microfluidic impedance-based flow cytometers have not yet or may never reach the extremely high throughput of conventional flow cytometers, the advantages of portability, simplicity, and ability to analyze single cells in small populations are, nevertheless, where chip-based cytometry can make a large impact. ' 2010International Society for Advancement of Cytometry Key terms microfluidics; impedance characterization; label free; single cell analysis; hydrodynamic focusing; sorting MICROFLUIDIC flow cytometers can bring many advantages to the field of flow cytometry (FCM). Compared to typical flow cytometry channel sizes, the miniaturized dimensions permit microfluidic systems to analyze single cells, to identify cellular variability in gene expression, or drug response within a cell population. Chipbased cytometers can have lower size and costs than conventional benchtop instruments, and may be portable. Today, the developmental aim for microfluidic systems is to reach the same sensitivity and capability for multiparametric analyses as delivered by conventional flow cytometers. Many efforts have been made to improve existing devices and to create new miniaturized high-end instruments. Microfluidic chips can incorporate on-chip cell preparation, cell culture, lysis, and modules for optical, electrophoretic, or genomic analysis (1). They are also suitable for analysis of cells in suspension as well as adherent cells.Miniaturized cytometry devices will have high impact in the development of point-of-care devices in developing countries. Accurate CD4 1 T-cell counts are used to monitor human immunodeficiency virus (HIV)-infected patients, and various thresholds of the number of CD4 1 T lymphocytes per ll of whole blood are used to start antiretroviral therapy (2-5). A simple, single-purpose CD4 cell counting device, which does not require standard laboratory equipment or trained laboratory personnel, could help some of the 33 million HIV-infected people worldwide monitor the stage of infection (6)(7)(8). In this application, increased analysis throughput is a secondary concern compared to increased sensitivity and specificity. Portable, miniatu...

With our adaptations it is possible to discriminate tandem conjugates of Cy5, Cy5.5, and Cy7 for eight-color immunophenotyping. Using this method, novel rare subsets of NK and NKT cells that are CD4/CD8 double positive are reported for the first time.

gamma-H2AX loss kinetics follows a bi-exponential decline with two definite decay times independent of LET. The higher contribution of the slow component determined for carbon ion exposure is thought to reflect the increased amount of complex DSB induced by high LET radiation.

Background: Polychromatic analysis of biological specimens has become increasingly important because of the emerging new fields of high-content and high-throughput single cell analysis for systems biology and cytomics. Combining different technologies and staining methods, multicolor analysis can be pushed forward to measure anything stainable in a cell. We term this approach hyperchromatic cytometry and present different components suitable for achieving this task. For cell analysis, slide based cytometry (SBC) technologies are ideal as, unlike flow cytometry, they are non-consumptive, i.e. the analyzed sample is fixed on the slide and can be reanalyzed following restaining of the object. Methods and Results: We demonstrate various approaches for hyperchromatic analysis on a SBC instrument, the Laser Scanning Cytometer. The different components demonstrated here include (1) polychromatic cytometry (staining of the specimen with eight or more different fluorochromes simultaneously), (2) iterative restaining (using the same fluorochrome for restaining and