Shlezinger Neta, Eizner Elad, Dubinchik Stas, Minz-Dub Anna, Tetroashvili Rachel, Reider Adi, Sharon Amir
Department of Molecular Biology and Ecology of Plants, Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel.
Department of Molecular Biology and Ecology of Plants, Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel. ; Department of Physical Electronics, Fleischman Faculty of Engineering, Tel Aviv University, Tel Aviv 69978, Israel.
Microb Cell. 2014 Nov 26;1(12):406-415. doi: 10.15698/mic2014.12.180.
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.
凋亡样程序性细胞死亡（A-PCD）是所有真核生物共有的普遍过程。由于与A-PCD相关的过程是保守的，因此可以通过一组标准标记来定义A-PCD。许多常用的测量A-PCD的方法都利用了在A-PCD过程中强度或细胞定位发生变化的荧光配体。在单细胞生物中，可以使用流式细胞仪通过对凋亡细胞的数量进行评分来量化A-PCD的水平。在多细胞生物中，由于组织的复杂性，A-PCD的量化更具问题。在丝状真菌中情况更为复杂，其中细胞核分布在不同的隔室中，每个隔室包含多个细胞核，这些细胞核也可以在隔室之间迁移。我们开发了SCAN，即细胞核计数与分析系统，并使用它根据两个标记——染色质浓缩和DNA链断裂来测量A-PCD。该软件包包括三个模块，用于对多核区域中的细胞核数量进行计数、对具有浓缩染色质的细胞核的相对数量进行评分以及计算具有DNA链断裂的细胞核的相对数量。与手动计数相比，该方法提供了相同或更好的结果，分析速度快，并且可以应用于大型数据集。虽然我们展示了该软件在测量真菌中A-PCD的实用性，但该方法很容易被用于测量其他类型多细胞标本中的A-PCD。
