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Proceedings Paper

Assessment of DNA replication in central nervous system by Laser Scanning Cytometry
Author(s): Dominik Lenz; Birgit Mosch; Jozsef Bocsi; Thomas Arendt; Attila Tárnok
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Paper Abstract

In neurons of patients with Alzheimers's disease (AD) signs of cell cycle re-entry as well as polyploidy have been reported, indicating that the entire or a part of the genome of the neurons is duplicated before its death but mitosis is not initiated so that the cellular DNA content remains tetraploid. It was concluded, that this imbalance is the direct cause of the neuronal loss in AD3. Manual counting of polyploidal cells is possible but time consuming and possibly statistically insufficient. The aim of this study was to develop an automated method that detects the neuronal DNA content abnormalities with Laser Scanning Cytometry (LSC). Frozen sections of formalin-fixed brain tissue of AD patients and control subjects were labelled with anti-cyclin B and anti-NeuN antibodies. Immunolabelling was performed using Cy5- and Cy2-conjugated secondary antibodies and biotin streptavidin or tyramid signal amplification. In the end sections of 20µm thickness were incubated with propidium iodide (PI) (50μg/ml) and covered on slides. For analysis by the LSC PI was used as trigger. Cells identified as neurons by NeuN expression were analyzed for cyclin B expression. Per specimen data of at least 10,000 neurons were acquired. In the frozen brain sections an automated quantification of the amount of nuclear DNA is possible with LSC. The DNA ploidy as well as the cell cycle distribution can be analyzed. A high number of neurons can be scanned and the duration of measuring is shorter than a manual examination. The amount of DNA is sufficiently represented by the PI fluorescence to be able to distinguish between eu- and polyploid neurons.

Paper Details

Date Published: 1 July 2004
PDF: 11 pages
Proc. SPIE 5322, Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues II, (1 July 2004); doi: 10.1117/12.528168
Show Author Affiliations
Dominik Lenz, Univ. Leipzig (Germany)
Birgit Mosch, Univ. Leipzig (Germany)
Jozsef Bocsi, Univ. Leipzig (Germany)
Thomas Arendt, Univ. Leipzig (Germany)
Attila Tárnok, Univ. Leipzig (Germany)

Published in SPIE Proceedings Vol. 5322:
Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues II
Dan V. Nicolau; Joerg Enderlein; Robert C. Leif; Daniel L. Farkas, Editor(s)

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