Abstract
The aim of this protocol is to provide a comprehensive description of the materials, equipment and reproducible methods to detect and analyze anaphase bridges in immunofluorescence microscopy using DAPI to detect cells that failed to completely segregate during mitosis. It describes the process of cell preparation, staining and microscopic settings for detection of anaphase bridges. The protocol has been adapted from our previous publication (Aschacher et al., 2016).
Keywords: Anaphase bridges, Cancer, DNA Damage
Background
During cell division it is vital for the maintenance of genome integrity that the genetic material is fully separated. For various reasons this process can be dysfunctional and as a result the sister chromatids are connected by DNA bridges, which most frequently happens during anaphase. Especially chromosomal fragile sites are associated with anaphase bridges (e.g., unprotected and unstable telomeres). Breakage, deletion, translocation non-disjunction and changes in chromosome number at these sites are often linked with cancer and other genetic diseases. Two types of anaphase bridges are described, the ultrafine DNA bridges, that cannot be detected by DAPI staining and the chromatin bridges, which are visualized by DAPI (Germann et al., 2014). The latter is described subsequently.This protocol describes a fast and simple method for the detection and calculation of anaphase bridges to provide an additional assay for telomere attrition in any publications.
Materials and Reagents
Equipment
Software
Procedure
Data analysis
The frequency of anaphase bridges is calculated as the ratio between cells exhibiting anaphase bridges and the total number of anaphase cells counting at least 100 anaphases. For example we distinguish for calculation i) all nuclei in all phases, ii) all cells in mitosis, iii) cells in anaphases, and iv) anaphase with bridges (also please find Figure 1b). For statistical significance Student's t-test values are calculated. Note: For this protocol, we treated tumour cell lines with scrambled siRNA or effective siRNA against LINE-1 mRNA as described previously (Aschacher et al., 2016). An effective LINE-1 siRNA treatment causes telomere attrition within three days and leads to anaphase bridges (Table 1 and Figure 2). Table 1. Statistical calculation for anaphase bridges. Representative table for counting of anaphase bridges is given. A minimum of three independent experiments should be done. Figure 2. Graphic example for statistical visualization of anaphase bridges. Counted nuclei (including interphase and mitosis), mitosis, anaphases and cells with anaphase bridges are given separately. Numbers of each count are given on the top of bars.
Recipes
Acknowledgments
This protocol was mainly adapted from the publication Aschacher et al. (2016). The work was supported by the Medical University of Vienna in the laboratories for surgical research.
References
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