Detection of Anaphase Bridge Formation by Immunofluorescence Microscopy in Mammalian Cells
采用免疫荧光显微技术检测哺乳动物细胞分裂后期桥的形成
发布: 2016年10月20日第6卷第20期 DOI: 10.21769/BioProtoc.1977 浏览次数: 12136
评审: HongLok LungAnonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
Jan 2016
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 (后期桥)
Cancer (癌症)
DNA Damage (DNA损伤)
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
- 8-chamber slides (e.g., NuncTM Lab-TekTM II chamber SlideTM system, Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 154534 )
- Nail polish (colourless, commercial available)
- Coverslips (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: Q10143263NR1 )
- Tumour cell line
Note: HCT-116 p53/wt was used in this protocol (ATCC, Germany). - Fetal bovine serum (FBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 26140079 )
- Dulbecco’s phosphate-buffered saline (PBS) (Sigma-Aldrich, catalog number: D8662 )
- Trypsin-EDTA solution, 0.25%, sterile-filtered (Sigma-Aldrich, catalog number: T4049 )
- McCoy’s 5A medium modified with sodium bicarbonate, without L-glutamine (Sigma-Aldrich, catalog number: M8403 )
- Formaldehyde solution (37%) (Sigma-Aldrich, catalog number: 252549 )
- Nuclear staining reagent (colour depends on secondary staining)
- 4’,6-diamidino-2-phenylindole (DAPI), colour blue (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 62247 )
- Hoechst 33342, trihydrochloride, trihydrate, colour blue (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: H3570 )
- DRAQ5TM fluorescent probe solution (5 mM), colour red (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 62251 )
- Propidium iodide (PI), colour red (Thermo Fisher Scientific, Molecular ProbesTM, catalog number: P21493 )
- Vectashield mounting medium (Vector Laboratories, USA)
- Triton X-100 (Sigma-Aldrich, catalog number: X100 )
- Growth medium (see Recipes)
- 3.7% formaldehyde solution (see Recipes)
- Permeabilization solution (see Recipes)
Equipment
- Cell culture microscope (e.g., Carl Zeiss, Vienna, Austria)
- Pipettes
- Hemocytometer (e.g., Bürker-Türk) (BRAND, catalog number: 719505 )
- Centrifuge (e.g., Eppendorf, Germany)
- 37 °C, 5% CO2 incubator
- Confocal laser scanning microscope (Carl Zeiss, model: LSM 800 )
Software
- Software for LSM800: ZEN lite (for free download find 'www.zeiss.com/microscopy/en_de/products/microscope-software/zen-lite.html')
Procedure
文章信息
版权信息
© 2016 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Aschacher, T. and Enzmann, F. (2016). Detection of Anaphase Bridge Formation by Immunofluorescence Microscopy in Mammalian Cells. Bio-protocol 6(20): e1977. DOI: 10.21769/BioProtoc.1977.
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分类
癌症生物学 > 基因组不稳定性及突变 > 细胞生物学试验
细胞生物学 > 细胞成像 > 荧光
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