Quantification of Colibactin-associated Genotoxicity in HeLa Cells by In Cell Western (ICW) Using γ-H2AX as a Marker
γ-H2AX作为标志物采用细胞内蛋白质印迹法（ICW）定量HeLa细胞中Colibactin相关基因毒性
发布: 2018年03月20日第8卷第6期 DOI: 10.21769/BioProtoc.2771 浏览次数: 7355
评审: Andrea PuharIsabel SilvaAnonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
Dec 2016
Abstract
The genotoxin colibactin is produced by several species of Enterobacteriaceae. This genotoxin induces DNA damage, cell cycle arrest, senescence and death in eukaryotic cells (Nougayrède et al., 2006; Taieb et al., 2016). Here we describe a method to quantify the genotoxicity of bacteria producing colibactin following a short infection of cultured mammalian cells with colibactin producing E. coli.
Keywords: Colibactin (Colibactin)
Infection (感染)
Escherichia coli (大肠埃希杆菌)
Genotoxin (基因毒素)
DNA damage (DNA损伤)
γ-H2AX (γ-H2AX)
Background
The genotoxin colibactin is a polyketide nonribosomal peptide hybrid compound produced by several species of Enterobacteriaceae. This toxin, synthesized by a machinery encoded on a 54 kb genomic locus, the pks island, induces DNA damages, cell cycle arrest, senescence and death in eukaryotic cells (Nougayrède et al., 2006; Taieb et al., 2016). The genotoxic activity of colibactin is dependent on a direct host cell-bacteria interaction and cannot be recapitulated from culture supernatant, killed bacteria, or bacterial lysates instead of life bacteria. Visualization and quantification of the colibactin genotoxic effect on eukaryotic cells can be assessed by quantification of the megalocytosis phenotype (for a protocol see Bossuet-Greif et al., 2017) or quantification of the double-strand DNA breaks in the host cell nucleus by a comet assay (revealing DNA fragmentation) or phosphorylation of the H2AX histone, a marker of double-strand DNA breaks. The phosphorylation of the histone H2AX is characterized as an early and sensitive reaction to genotoxic agents (Audebert et al., 2010). H2AX phosphorylation was demonstrated to be 10-100 times more sensitive than the comet assay in vitro as well as in vivo (Audebert et al., 2010). The quantification of phosphorylated histone H2AX (γ-H2AX) can be processed by the In-Cell Western Assay, an immunochemical assay that uses fluorescence to detect and quantify proteins in fixed cells (Audebert et al., 2010). Here we describe an adapted assay allowing the measurement of γ-H2AX in 96-well plate using In-Cell Western, following a short infection of cultured mammalian cells with colibactin-producing bacteria (Martin et al., 2013; Bossuet-Greif et al., 2016; Tronnet et al., 2017).
Materials and Reagents
- Black tissue culture plate 96 wells flat bottom (Greiner Bio One International, catalog number: 655090 )
- Parafilm
- Aluminum foil
- pks+ and pks- Escherichia coli strains (stored in LB 20% glycerol at -80 °C)
Note: Strains typically used as positive controls in the authors’ laboratory are probiotic strain Nissle 1917 or the commensal strain M1/5. Strains used as a negative control are the K-12 strain MG1655. Our lab can provide these strains. - HeLa cells (ATCC, catalog number: CCL-2 ), 20 passages maximum
- Lennox L broth base (LB medium; Thermo Fisher Scientific, InvitrogenTM, catalog number: 12780029 )
- Dulbecco’s modified Eagle medium (DMEM) with 25 mM HEPES (Thermo Fisher Scientific, GibcoTM, catalog number: 42430 )
- Hanks’ balanced salt solution (HBSS; Sigma-Aldrich, catalog number: H8264 )
- Gentamicin solution 50 mg/ml (Sigma-Aldrich, catalog number: G1397 )
- Dulbecco’s phosphate buffered saline (PBS; Sigma-Aldrich, catalog number: D8537 )
- Dulbecco’s modified Eagle medium (DMEM), high glucose, GlutaMax Supplement, pyruvate (Thermo Fisher Scientific, GibcoTM, catalog number: 31966021 )
- Fetal bovine serum (FBS; Thermo Fisher Scientific, GibcoTM, catalog number: 10270106 )
- Non-essential amino acids solution (NEAA) 100x (Thermo Fisher Scientific, GibcoTM, catalog number: 11140035 )
- Paraformaldehyde (PFA) 20% (Electron Microscopy Sciences, catalog number: 15713 )
- 10x PBS (Sigma-Aldrich, catalog number: D1408 )
- Ammonium chloride (NH4Cl; BioUltra, Sigma-Aldrich, catalog number: 09718-250G )
- TritonTM X-100 (Sigma-Aldrich, catalog number: X100-500ML )
- MAXblock blocking medium (Active Motif, catalog number: 15252 )
- Phosphatase inhibitor PHOSTOP (10x, Roche Diagnostics, catalog number: 04906837001 )
- RNase (Sigma-Aldrich, catalog number: R6513 )
- Sodium chloride (NaCl; Sigma-Aldrich, catalog number: S7653 )
- Sodium azide (Sigma-Aldrich, catalog number: S8032 )
- Rabbit monoclonal anti-γ-H2AX antibody (Cell Signaling Technology, catalog number: 9718 )
- IRDyeTM 800CW-conjugated goat anti-rabbit secondary antibody (2 mg/ml, Biotium (distributor Interchim), catalog number: 20078 )
- RedDotTM2 (200x in DMSO, Biotium (distributor Interchim), catalog number: 40061 )
- HeLa cell culture medium (see Recipes)
- Fixation solution (see Recipes)
- Neutralization solution (see Recipes)
- 10% Triton X-100 (see Recipes)
- Permeabilization solution (see Recipes)
- Blocking solution (see Recipes)
- Permeabilization solution (see Recipes)
- PST buffer (see Recipes)
- 100x azide (see Recipes)
- Anti-γ-H2AX solution (see Recipes)
- Secondary antibody solution (see Recipes)
Equipment
- 37 °C, 5% CO2 incubator for cell cultures
- 37 °C incubator for bacterial cultures
- Microplate reader with 680 and 800 nm channels (here, Odyssey Infrared Imaging Scanner, Li-Cor ScienceTec, Les Ulis, France)
- Microplate reader for absorbance measurement at 600 nm (TECAN Infinite Pro)
- Colorimeter to measure the absorbance at 600 nm of bacterial cultures (Biochrom, model: WPA CO7500 )
- Variable Speed Rocker (VWR, catalog number: 75832-308 )
- Chemical safety hood
- Clean bench
- Inverted microscope (Olympus, model: CKX31 )
- Pipettes and multichannel micropipette 30-300 µl
Software
- GraphPad Prism 6.0
Procedure
文章信息
版权信息
© 2018 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Tronnet, S. and Oswald, E. (2018). Quantification of Colibactin-associated Genotoxicity in HeLa Cells by In Cell Western (ICW) Using γ-H2AX as a Marker. Bio-protocol 8(6): e2771. DOI: 10.21769/BioProtoc.2771.
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分类
微生物学 > 微生物-宿主相互作用 > 体外实验模型
细胞生物学 > 细胞染色 > 蛋白质
生物化学 > 蛋白质 > 荧光
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