Measurement of apoptosis by SCAN©, a system for counting and analysis of fluorescently labelled nuclei

Authors:

Neta Shlezinger1,#, Elad Eizner1,2,#, Stas Dubinchik2, Anna Minz-Dub1, Rachel Tetroashvili1, Adi Reider1, Amir Sharon1

doi: 10.15698/mic2014.12.180
Volume 1, pp. 406 to 415, published 26/11/2014.

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Affiliations:

1 Department of Molecular Biology and Ecology of Plants, Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel.

2 Department of Physical Electronics, Fleischman Faculty of Engineering, Tel Aviv University, Tel Aviv 69978, Israel.

# Equal contribution.

Keywords: 

apoptosis, PCD, fungi, DNA condensation, TUNEL.

Abbreviations:

A-PCD - apoptosis-like programmed cell death,

FACS - fluorescence activated flow cytometry,

TUNEL - terminal dUTP nick end-labeling,

DAPI - 4',6-diamidino-2-phenylindole.

Corresponding Author(s):

Amir Sharon, Department of Molecular Biology and Ecology of Plants, Faculty of Life Sciences Tel Aviv University; Tel Aviv 69978, Israel amirsh@ex.tau.ac.il

Conflict of interest statement:

The authors declare no conflict of interest.

Please cite this article as:

Neta Shlezinger, Elad Eizner, Stas Dubinchik, Anna Minz-Dub, Rachel Tetroashvili, Adi Reider, Amir Sharon (2014). Measurement of apoptosis by SCAN©, a system for counting and analysis of fluorescently labelled nuclei. Microbial Cell 1(12): 406-415.

© 2014 Shlezinger et al. This is an open-access article released under the terms of the Creative Commons Attribution (CC BY) license, which allows the unrestricted use, distribution, and reproduction in any medium, provided the original author and source are acknowledged.

Abstract:

Apoptosis-like programmed cell death (A-PCD) is a universal process common to all types of eukaryotic organisms. Because A-PCD-associated processes are conserved, it is possible to define A-PCD by a standard set of markers. Many of the popular methods to measure A-PCD make use of fluorescent ligands that change in intensity or cellular localization during A-PCD. In single cell organisms, it is possible to quantify levels of A-PCD by scoring the number of apoptotic cells using flow cytometry instruments. In a multicellular organism, quantification of A-PCD is more problematic due to the complex nature of the tissue. The situation is further complicated in filamentous fungi, in which nuclei are divided between compartments, each containing a number of nuclei, which can also migrate between the compartments. We developed SCAN©, a System for Counting and Analysis of Nuclei, and used it to measure A-PCD according to two markers – chromatin condensation and DNA strand breaks. The package includes three modules designed for counting the number of nuclei in multi-nucleated domains, scoring the relative number of nuclei with condensed chromatin, and calculating the relative number of nuclei with DNA strand breaks. The method provides equal or better results compared with manual counting, the analysis is fast and can be applied on large data sets. While we demonstrated the utility of the software for measurement of A-PCD in fungi, the method is readily adopted for measurement of A-PCD in other types of multicellular specimens.