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   

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Pathogens and Disease
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

  1. Black tissue culture plate 96 wells flat bottom (Greiner Bio One International, catalog number: 655090 )
  2. Parafilm
  3. Aluminum foil
  4. 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.
  5. HeLa cells (ATCC, catalog number: CCL-2 ), 20 passages maximum
  6. Lennox L broth base (LB medium; Thermo Fisher Scientific, InvitrogenTM, catalog number: 12780029 )
  7. Dulbecco’s modified Eagle medium (DMEM) with 25 mM HEPES (Thermo Fisher Scientific, GibcoTM, catalog number: 42430 )
  8. Hanks’ balanced salt solution (HBSS; Sigma-Aldrich, catalog number: H8264 )
  9. Gentamicin solution 50 mg/ml (Sigma-Aldrich, catalog number: G1397 )
  10. Dulbecco’s phosphate buffered saline (PBS; Sigma-Aldrich, catalog number: D8537 )
  11. Dulbecco’s modified Eagle medium (DMEM), high glucose, GlutaMax Supplement, pyruvate (Thermo Fisher Scientific, GibcoTM, catalog number: 31966021 )
  12. Fetal bovine serum (FBS; Thermo Fisher Scientific, GibcoTM, catalog number: 10270106 )
  13. Non-essential amino acids solution (NEAA) 100x (Thermo Fisher Scientific, GibcoTM, catalog number: 11140035 )
  14. Paraformaldehyde (PFA) 20% (Electron Microscopy Sciences, catalog number: 15713 )
  15. 10x PBS (Sigma-Aldrich, catalog number: D1408 )
  16. Ammonium chloride (NH4Cl; BioUltra, Sigma-Aldrich, catalog number: 09718-250G )
  17. TritonTM X-100 (Sigma-Aldrich, catalog number: X100-500ML )
  18. MAXblock blocking medium (Active Motif, catalog number: 15252 )
  19. Phosphatase inhibitor PHOSTOP (10x, Roche Diagnostics, catalog number: 04906837001 )
  20. RNase (Sigma-Aldrich, catalog number: R6513 )
  21. Sodium chloride (NaCl; Sigma-Aldrich, catalog number: S7653 )
  22. Sodium azide (Sigma-Aldrich, catalog number: S8032 )
  23. Rabbit monoclonal anti-γ-H2AX antibody (Cell Signaling Technology, catalog number: 9718 )
  24. IRDyeTM 800CW-conjugated goat anti-rabbit secondary antibody (2 mg/ml, Biotium (distributor Interchim), catalog number: 20078 )
  25. RedDotTM2 (200x in DMSO, Biotium (distributor Interchim), catalog number: 40061 )
  26. HeLa cell culture medium (see Recipes)
  27. Fixation solution (see Recipes)
  28. Neutralization solution (see Recipes)
  29. 10% Triton X-100 (see Recipes)
  30. Permeabilization solution (see Recipes)
  31. Blocking solution (see Recipes)
  32. Permeabilization solution (see Recipes)
  33. PST buffer (see Recipes)
  34. 100x azide (see Recipes)
  35. Anti-γ-H2AX solution (see Recipes)
  36. Secondary antibody solution (see Recipes)

Equipment

  1. 37 °C, 5% CO2 incubator for cell cultures
  2. 37 °C incubator for bacterial cultures
  3. Microplate reader with 680 and 800 nm channels (here, Odyssey Infrared Imaging Scanner, Li-Cor ScienceTec, Les Ulis, France)
  4. Microplate reader for absorbance measurement at 600 nm (TECAN Infinite Pro)
  5. Colorimeter to measure the absorbance at 600 nm of bacterial cultures (Biochrom, model: WPA CO7500 )
  6. Variable Speed Rocker (VWR, catalog number: 75832-308 )
  7. Chemical safety hood
  8. Clean bench
  9. Inverted microscope (Olympus, model: CKX31 )
  10. Pipettes and multichannel micropipette 30-300 µl

Software

  1. GraphPad Prism 6.0

Procedure

Day 1

  1. Bacteria are cultured overnight in 3 ml of LB from -80 °C glycerol stock or a colony on an LB agar plate at 37 °C with shaking (220 rpm).
  2. HeLa cells are dispensed in a black 96-well tissue culture plate with transparent bottom (1.5 x 104 cells/well) and grown for 24 h in 200 μl HeLa cell culture medium (see Recipes) at 37 °C in a CO2 incubator.
    Note: Importantly, don’t dispense any cells in the two bottom-left wells (see Figure 1: plate infection scheme).


    Figure 1. Schematic representation of plate infection example. Bacteria are inoculated in the two first rows in duplicate (in 200 μl/well of DMEM-HEPES medium), at an MOI of 50 and then 2-fold serially diluted (by successively transferring 100 μl to the rows below). The final volume is 100 μl. The left column is kept for the controls (red wells column).

Day 2

  1. Bacterial preparation
    Bacteria are sub-cultured by diluting 50 µl of the overnight culture in 5 ml of DMEM-HEPES medium (i.e., 1:100 dilution) and grown at 37 °C with agitation (220 rpm) until an average OD600nm = 0.6. The number of bacteria per ml is determined by measuring the OD600nm of 1 ml of the culture.
    Note: The typical value for E. coli is 1 unit of the OD600nm corresponds to 5 x 108 bacteria/ml.
  2. HeLa cells preparation
    1. Before infection, eukaryotic cells are cautiously washed once with pre-warmed (37 °C) HBSS.
    2. 100 μl of DMEM-HEPES medium is added in all the wells, except the 2 first rows, corresponding to the highest number of bacteria per cell (maximum multiplicity of infection or MOI) where 200 μl of DMEM-HEPES medium is added (Figure 1).
  3. Infection assay
    1. After determination of the multiplicity of infection (MOI = number of bacteria per HeLa cell at the onset of the infection), the appropriate number of bacteria is added in the first 2 rows of the plate (containing 200 μl DMEM-HEPES medium).
    2. 2-fold serial dilution is performed to create an infectious dose-effect, in a final DMEM-HEPES medium volume of 100 μl (Figure 1).
      Note: As an example, for an MOI of 50, add 50 x 1.5 x 104 = 75 x 104 bacteria for a well containing 1.5 x 104 HeLa cells. The number of dispensed cells is taken into account for the MOI calculation.
    3. The plate is incubated for 4 h in a cell culture incubator.
  4. End of the infection and post-infection incubation
    1. After infection of 4 h, the absorbance at 600 nm is measured with a microplate reader, to monitor bacterial growth. The typical values obtained are around 0.2-0.3 for MOI 50.
    2. Then, cells are washed carefully at least 3 times with an increasing volume of pre-warm (37 °C) HBSS (100 μl, 150 μl and finally 200 μl). The plate is observed under an inverted microscope to check whether most of the bacteria are removed. If required, additional washes can be performed.
    3. Finally, cells are incubated in 200 μl cell culture medium (see Recipes) for 3 h with 200 mg/ml gentamicin in a cell culture incubator.
  5. Cells fixation
    1. The cells are washed carefully in cold PBS (100 μl) three times.
    2. 100 μl fixation solution (see Recipes) is added for 20 min under a chemical safety hood.
  6. Paraformaldehyde neutralization
    1. After the fixation step, cells are washed with PBS (100 μl per well), for 5 min under rapid agitation (variable speed shaker, 20-25 rpm). This washing step is repeated 3 times.
    2. Then, the paraformaldehyde is neutralized by adding 50 μl of neutralization solution (see Recipes) for 2 min under slow agitation (3-5 rpm).
  7. Cells permeabilization
    1. The cells are washed 3 times for 5 min with PBS, 100 μl per well, under rapid agitation (20-25 rpm) as previously (Step 6).
    2. Then cells are permeabilized with cold permeabilization solution (see Recipes), 50 μl per well for 5 min, under slow agitation.
  8. Blocking step
    1. Cells are washed 3 times with PST buffer (see Recipes), 100 μl per well, under agitation (20-25 rpm).
    2. Then, 50 μl/well of blocking solution (see Recipes) is added, and the plate is incubated at room temperature for 60 min under agitation (3-5 rpm).
  9. Anti-γ-H2AX immunostaining step
    1. Before immunostaining, cells are washed with PST buffer 3 times, 100 μl for 5 min under agitation (20-25 rpm).
    2. 25 μl per well of anti-γ-H2AX solution (see Recipes) is added and the plate is incubated for 2 h at room temperature or overnight at 4 °C (in that case, the plate has to be sealed carefully with Parafilm to avoid drying), under agitation (3-5 rpm).
      Note: This solution is not added in the controls A and C (Figure 1), 25 μl of PST buffer is added instead during the incubation time.
  10. Secondary detection step
    1. Cells are first washed 3 times with PST buffer, 100 μl per well for 5 min under agitation (20-25 rpm).
    2. Secondary detection is carried out using an infrared fluorescent dye conjugated IRDyeTM 800CW-conjugated goat anti-rabbit antibodies absorbing at 800 nm (1/1,000) in PST buffer. For DNA labeling, 1/1,000 dilution of RedDotTM2 in PST is used together with the secondary antibody (see secondary antibody solution in Recipes), 25 μl per well in every well of the plate.
    3. The plate is incubated in the dark for 1 h under agitation (3-5 rpm).
  11. Plate scanning step
    1. The plate is washed 3 times in PST buffer, 100 μl per well for 5 min under agitation (20-25 rpm) in obscurity (covered with aluminum foil).
    2. Then, the plate is dried by reversing and patting it delicately on a tissue.
    3. The DNA and the γ-H2AX were simultaneously visualized using an Odyssey Infrared Imaging Scanner with the 680 nm fluorophore (red color) and the 800 nm fluorophore (green dye), respectively (Figure 3A).
    Note: Once the plate dried (Step 11b), it can be conserved several months, at 4 °C protected from the light, upside down, before visualization.

Data analysis

A quantitative analysis of the frequency of DNA double-strand breaks can be performed using the fluorescence values (in RFU) obtained from the scans (Figures 2 and 3B). Firstly, the average γ-H2AX fluorescence per cell is obtained by dividing the measured γ-H2AX fluorescence per well (at 800 nm) by the corresponding measured fluorescence for RedDot-labelled DNA per well (at 680 nm) (Figure 2). The value for γ-H2AX fluorescence per cell is divided by the average fluorescence value at 800 nm obtained for the two wells serving as vehicle control (here corresponding to the wells marked ‘C’ in Figures 1 and 2) to determine the percent change in phosphorylation of H2AX levels relative to the vehicle control. The obtained number is defined as a genotoxic index (or γ-H2AX fold induction) and typically ranges from 0 to 2 in completely healthy cells, to 5 to 7 in cells infected with colibactin-producing E. coli at MOI 25 (Figure 2 and Figure 3B).


Figure 2. Data analysis of γ-H2AX quantification. Representative image of how we process data from the raw numbers to the calculation of the γ-H2AX fold induction (genotoxic index).


Figure 3. Quantification of phosphorylated H2AX (γ-H2AX) from infected HeLa cells with pks+ E. coli or with pks- E. coli at various MOI. A. Example of an In-Cell Western plate image with merged detection of total DNA (red, 680 nm) and γ-H2AX (green, 800 nm). A high level of γ-H2AX can be observed at high MOI for the cells infected with E. coli pks+. B. The γ-H2AX fold induction (γ-H2AX fluorescence normalized to the amount of cells per well), calculated relative to the control (non-infected cells), reveals a genotoxic dose-response depending on the MOI in cells infected by colibactin-producing bacteria (E. coli pks+) compared to cells infected by non colibactin-producing bacteria (E. coli pks-). Data represented in the graph were obtained from two biological replicates and two independent experiments. Data were plotted using GraphPad Prism 6.0. The mean with standard deviation (sd) is shown.

Recipes

  1. HeLa cells culture medium
    450 ml DMEM high glucose with pyruvate and GlutaMax
    50 ml fetal bovine serum
    5 ml NEAA
  2. Fixation solution (1x PBS, 4% PFA)
    5 ml PFA stock solution
    5 ml 20% 10x PBS
    40 ml distilled water
    Aliquot and store at -20 °C
    Defreeze one aliquot the day of the experiment
  3. Neutralization solution (20 mM NH4Cl in PBS)
    a. Prepare the stock solution (1 M NH4Cl) in distilled water and store at 4 °C
    b. Dissolve the stock solution in PBS to reach the appropriate concentration
    c. Keep on ice until use
  4. 10% Triton X-100
    500 μl Triton X-100 in 50 ml PBS
    This solution can be stored at 4 °C for 2-3 weeks
  5. Permeabilization solution (1x PBS, 0.2% Triton X-100)
    200 μl 10% Triton X-100
    10 ml PBS
    Keep on ice until use
  6. Blocking solution (MAXblock blocking medium, 1x PHOSTOP inhibitor, RNase [100 μg/ml])
    4.5 ml MAXblock blocking medium
    500 μl 10x PHOSTOP inhibitor
    50 μl RNase stock solution (10 mg/ml in PBS, 10 mM HEPES pH 7.5, 15 μM NaCl, aliquot stored at -20 °C)
    Keep on ice until use
  7. PST buffer (PBS, 2% fetal calf serum, 0.2% Triton X-100)
    48 ml PBS
    1 ml 2% fetal calf serum
    1 ml 0.2% Triton X-100 in PBS
  8. 100x azide
    Dissolve sodium azide in distilled water at a final concentration of 50 mg/ml (5% w/w), store at 4 °C
  9. Anti-γ-H2AX solution (rabbit monoclonal anti-γ-H2AX [1/200] in PST buffer, 500 μg/ml azide)
    1.5 ml PST buffer
    7.5 μl anti-γ-H2AX
    15 μl 100x azide
    Note: This solution can be reused 2-3 times, and stored at 5 °C for several weeks.
  10. Secondary antibody solution (IRDyeTM 800CW Goat Anti-Rabbit [1/1,000], RedDotTM2 [1/1,000] in PST buffer)
    2.5 ml PST
    2.5 μl IRDyeTM 800CW Goat Anti-Rabbit
    2.5 μl RedDotTM2

Acknowledgments

This protocol was adapted from previously published work (Audebert et al., 2010; Martin et al., 2013; Bossuet-Greif et al., 2016; Tronnet et al., 2017). This work was supported by the Agence Nationale de la Recherche (France) [grant ANR-13-BSV3–0015-02, ANR-13-BSV1–0028-01]. The authors declare no conflicts of interest or competing interests, regarding the publication of this protocol.

References

  1. Audebert, M. Riu, A., Jacques, C., Hillenweck, A., Jamin, E. L., Zalko, D., Cravedi, J. P. (2010). Use of the gammaH2AX assay for assessing the genotoxicity of polycyclic aromatic hydrocarbons in human cell lines. Toxicol Lett 199: 182-192.
  2. Bossuet-Greif, N., Belloy, M., Boury, M., Oswald, E. and Nougayrede, J. (2017). Protocol for HeLa cells infection with Escherichia coli strains producing colibactin and quantification of the induced DNA-damage. Bio-protocol 7(16): e2520.
  3. Bossuet-Greif, N., Dubois, D., Petit, C., Tronnet, S., Martin, P., Bonnet, R., Oswald, E. and Nougayrede, J. P. (2016). Escherichia coli ClbS is a colibactin resistance protein. Mol Microbiol 99(5): 897-908.
  4. Martin, P., Marcq, I., Magistro, G., Penary, M., Garcie, C., Payros, D., Boury, M., Olier, M., Nougayrede, J. P., Audebert, M., Chalut, C., Schubert, S. and Oswald, E. (2013). Interplay between siderophores and colibactin genotoxin biosynthetic pathways in Escherichia coli. PLoS Pathog 9(7): e1003437.
  5. Nougayrède, J. P., Homburg, S., Taieb, F., Boury, M., Brzuszkiewicz, E., Gottschalk, G., Buchrieser, C., Hacker, J., Dobrindt, U. and Oswald, E. (2006). Escherichia coli induces DNA double-strand breaks in eukaryotic cells. Science 313(5788): 848-851.
  6. Taieb, F., Petit, C., Nougayrede, J. P. and Oswald, E. (2016). The enterobacterial genotoxins: cytolethal distending toxin and colibactin. EcoSal Plus 7(1).
  7. Tronnet, S., Garcie, C., Brachmann, A. O., Piel, J., Oswald, E. and Martin, P. (2017). High iron supply inhibits the synthesis of the genotoxin colibactin by pathogenic Escherichia coli through a non-canonical Fur/RyhB-mediated pathway. Pathog Dis 75(5).

简介

基因毒素colibactin由几种肠杆菌科产生。 该基因毒素在真核细胞中诱导DNA损伤,细胞周期停滞,衰老和死亡(Nougayrède等人,2006; Taieb等人,2016)。 在这里,我们描述了一种方法来量化生产colibactin的细菌的基因毒性,在短时间感染培养的哺乳动物细胞后,产生e colibactin。大肠杆菌。

【背景】基因毒素colibactin是由几种肠杆菌科产生的聚酮化合物非核糖体肽杂交化合物。由编码在54kb基因组基因座pks岛上的机器合成的这种毒素在真核细胞中诱导DNA损伤,细胞周期停滞,衰老和死亡(Nougayrède等人, 2006年; Taieb 等人,2016年)。大肠杆菌素的基因毒性活性取决于直接的宿主细胞 - 细菌相互作用,并且不能从培养物上清液,杀死的细菌或细菌裂解物而非生命细菌重演。可以通过量化巨细胞病表型(对于方案参见Bossuet-Greif等人,2017)或双链DNA断裂的定量来评估对真核细胞的大肠杆菌素基因毒性效应的可视化和定量在彗星试验(揭示DNA片段化)或H2AX组蛋白磷酸化作用宿主细胞核中,双链DNA标记断裂。组蛋白H2AX的磷酸化表征为对遗传毒性剂的早期和敏感反应(Audebert等人,2010)。证明H2AX磷酸化比彗星试验在体外以及体内灵敏度高10-100倍(Audebert等人, 2010)。磷酸化组蛋白H2AX(γ-γ-H2AX)的定量可以通过In-Cell Western Assay进行处理,In-Cell Western Assay是使用荧光检测和定量固定细胞中的蛋白质的免疫化学测定法(Audebert等人。,2010)。在此我们描述了一种适应性测定法,其允许使用In-Cell Western在96孔板中用产生colibactin的细菌短时间感染培养的哺乳动物细胞来测量γ-γ-H2AX(Martin等人,2013; Bossuet-Greif等人,2016; Tronnet等人,,2017)。

关键字:Colibactin, 感染, 大肠埃希杆菌, 基因毒素, DNA损伤, γ-H2AX

材料和试剂

  1. 黑色组织培养板96孔平底(Greiner Bio One International,目录号:655090)
  2. Parafilm
  3. 铝箔
  4. + 和 pks - 大肠杆菌菌株(储存在-80℃的LB 20%甘油中)
    注:在作者的实验室中通常用作阳性对照的菌株是益生菌菌株Nissle 1917或共生菌株M1 / 5。用作阴性对照的菌株是K-12菌株MG1655。我们的实验室可提供这些菌株。
  5. HeLa细胞(ATCC,目录号:CCL-2),最多20代传代
  6. Lennox L肉汤培养基(LB培养基; Thermo Fisher Scientific,Invitrogen TM,目录号:12780029)
  7. 带有25mM HEPES(Thermo Fisher Scientific,Gibco TM,目录号:42430)的Dulbecco's改良伊格尔培养基(DMEM)
  8. Hanks平衡盐溶液(HBSS; Sigma-Aldrich,目录号:H8264)
  9. 庆大霉素溶液50mg / ml(Sigma-Aldrich,目录号:G1397)
  10. Dulbecco's磷酸盐缓冲盐水(PBS; Sigma-Aldrich,目录号:D8537)
  11. Dulbecco's改良的Eagle培养基(DMEM),高葡萄糖,GlutaMax补充物,丙酮酸盐(Thermo Fisher Scientific,Gibco TM,产品目录号:31966021)
  12. 胎牛血清(FBS; Thermo Fisher Scientific,Gibco TM,目录号:10270106)
  13. 非必需氨基酸溶液(NEAA)100x(Thermo Fisher Scientific,Gibco TM,产品目录号:11140035)

  14. 多聚甲醛(PFA)20%(Electron Microscopy Sciences,目录号:15713)
  15. 10x PBS(Sigma-Aldrich,目录号:D1408)
  16. 氯化铵(NH4Cl; BioUltra,Sigma-Aldrich,目录号:09718-250G)
  17. Triton TM X-100(Sigma-Aldrich,目录号:X100-500ML)
  18. MAXblock阻塞介质(Active Motif,目录号:15252)
  19. 磷酸酶抑制剂PHOSTOP(10倍,Roche Diagnostics,目录号:04906837001)
  20. RNase(Sigma-Aldrich,目录号:R6513)
  21. 氯化钠(NaCl; Sigma-Aldrich,目录号:S7653)
  22. 叠氮化钠(Sigma-Aldrich,目录号:S8032)
  23. 兔单克隆抗γ-em2AX抗体(Cell Signaling Technology,目录号:9718)
  24. IRDye TM 800CW缀合的山羊抗兔二抗(2mg / ml,Biotium(经销商Interchim),目录号:20078)
  25. RedDot TM 2(200x在DMSO中,Biotium(分销商Interchim),目录号:40061)
  26. HeLa细胞培养基(见食谱)
  27. 固定解决方案(请参阅食谱)
  28. 中和解决方案(请参阅食谱)
  29. 10%Triton X-100(见食谱)
  30. 透化溶液(见食谱)
  31. 阻止解决方案(请参阅食谱)
  32. 透化溶液(见食谱)
  33. PST缓冲区(请参阅食谱)
  34. 100倍叠氮化物(见食谱)
  35. Anti-em -H2AX解决方案(请参阅食谱)
  36. 二抗(见配方)

设备

  1. 37°C,5%CO 2培养箱用于细胞培养
  2. 37°C培养箱用于细菌培养
  3. 具有680和800 nm通道的酶标仪(这里是Odyssey红外成像扫描仪,Li-Cor ScienceTec,法国Les Ulis)
  4. 用于600 nm吸光度测量的酶标仪(TECAN Infinite Pro)
  5. 用于测量细菌培养物在600nm处的吸光度的比色计(Biochrom,型号:WPA CO7500)
  6. 变速摇臂(VWR,目录号:75832-308)
  7. 化学安全罩
  8. 洁净工作台
  9. 倒置显微镜(奥林巴斯,型号:CKX31)
  10. 移液器和多通道微量移液器30-300微升

软件

  1. GraphPad Prism 6.0

程序

第1天

  1. 将细菌在3ml LB中从-80℃甘油贮存液中或在LB琼脂平板上的菌落在37℃下振荡(220rpm)培养过夜。
  2. 将HeLa细胞分配在具有透明底部(1.5×10 4个细胞/孔)的黑色96孔组织培养板中并在37℃在200μlHeLa细胞培养基中生长24小时(参见食谱) °C在CO 2培养箱中培养。
    注意:重要的是,不要在左下两个孔中分配任何细胞(见图1:平板感染方案)。


    图1.平板感染实例的示意图。细菌以两份第一行接种(一式两份,以200μl/孔的DMEM-HEPES培养基),MOI为50,然后2倍连续稀释(通过连续转移100μl到下面的行)。最终体积为100μl。
    左栏保存为控件(红色的井栏)。

第2天

  1. 细菌制剂
    通过在5ml DMEM-HEPES培养基(即1:100稀释)中稀释50μl过夜培养物来细菌培养,并在37℃搅拌(220rpm)下培养直至平均OD 600nm = 0.6。通过测量1ml培养物的OD 600nm来确定每ml细菌的数量。
    注意:大肠杆菌的典型值是1单位的OD 600nm对应于5×10 8 8×10 6个细胞。 /细菌/ ml。
  2. HeLa细胞制备
    1. 在感染之前,用预热(37℃)HBSS仔细洗涤真核细胞一次。
    2. 在所有孔中加入100μlDMEM-HEPES培养基,除了第2行外,对应于每孔加入200μlDMEM-HEPES培养基的最高细菌数量(最大感染复数或MOI)(图1 )。
  3. 感染测定
    1. 确定感染复数(MOI =感染开始时每HeLa细胞的细菌数量)后,将适当数量的细菌加入平板的前2行(含有200μlDMEM-HEPES培养基)中。 br />
    2. 进行2倍系列稀释以产生感染剂量效应,在100μl的最终DMEM-HEPES培养基体积中(图1)。
      注:例如,对于50的MOI,添加50 x 1.5 x 10 4 = 75 x 10 对于含有1.5×10 4 HeLa的孔的细菌来说,细胞。 MOI计算中会考虑分配的单元格数量。
    3. 将培养板在细胞培养箱中培养4小时。
  4. 感染结束和感染后孵化
    1. 感染4小时后,用酶标仪测量600nm处的吸光度,以监测细菌生长。 MOI 50的典型值为0.2-0.3。
    2. 然后,用增加体积的预热(37℃)HBSS(100μl,150μl,最后200μl)仔细洗涤细胞至少3次。在倒置显微镜下观察平板以检查大部分细菌是否被除去。如果需要,可以执行额外的清洗。
    3. 最后,将细胞在200μl细胞培养基(参见配方)中用200mg / ml庆大霉素在细胞培养箱中孵育3小时。
  5. 细胞固定
    1. 细胞在冷的PBS(100μl)中仔细洗涤三次。
    2. 在化学安全罩下加入100μl固定溶液(参见食谱)20分钟。
  6. 多聚甲醛中和
    1. 固定步骤后,在快速搅拌(变速振荡器,20-25rpm)下用PBS(每孔100μl)洗涤细胞5分钟。该洗涤步骤重复3次。
    2. 然后,通过在缓慢搅拌(3-5rpm)下加入50μl中和溶液(参见配方)2分钟来中和多聚甲醛。
  7. 细胞透化
    1. 如前所述(步骤6),在快速搅拌(20-25rpm)下用PBS,每孔100μl洗涤细胞3次,每次5分钟。
    2. 然后在缓慢搅拌下用冷透化溶液使细胞透化(参见食谱),每孔50μl,5分钟。
  8. 阻止步骤

    1. 在搅拌下(20-25rpm),用PST缓冲液(参见食谱)将细胞洗涤3次,每孔100μl。
    2. 然后,加入50μl/孔的封闭溶液(参见配方),并将板在搅拌(3-5rpm)下在室温下温育60分钟。
  9. 抗-γ-H2AX免疫染色步骤
    1. 在免疫染色之前,用PST缓冲液洗涤细胞3次,100μl在搅拌(20-25rpm)下5分钟。
    2. 每孔加入25μl抗-γH2-H2AX溶液(参见配方)并将板在室温下温育2小时或在4℃下过夜(在这种情况下,板必须小心地用Parafilm密封以避免干燥),在搅拌下(3-5rpm)。
      注意:这个解决方案并不添加到对照A和C(图1)中,而是在孵育时间内添加25μl的PST缓冲液。
  10. 次要检测步骤
    1. 首先用PST缓冲液将细胞洗涤3次,每孔100μl在搅拌(20-25rpm)下5分钟。
    2. 使用在PST缓冲液中吸收800nm(1 / 1,000)的红外荧光染料缀合的IRDye TM 800CW缀合的山羊抗兔抗体进行二次检测。对于DNA标记,将PST中的1 / 1,000稀释的RedDot TM TM2与二抗一起使用(参见配方中的二抗溶液),在每个孔中每孔25μl。

    3. 在搅拌下(3-5rpm)将板在黑暗中孵育1小时
  11. 平板扫描步骤
    1. 将板在PST缓冲液中洗涤3次,每孔100μl,在搅拌下(20-25rpm)在模糊(用铝箔覆盖)下处理5分钟。
    2. 然后,通过在组织上微调并轻轻拍打来干燥平板。
    3. 使用具有680nm荧光团(红色)和800nm荧光团(绿色染料)的奥德赛红外成像扫描仪分别使DNA和γ-H2AX可视化(图3A)。
    注意:一旦板干燥(步骤11b),它可以保存好几个月,在4°C避光,颠倒,在可视化之前。

数据分析

可以使用从扫描获得的荧光值(以RFU计)进行DNA双链断裂频率的定量分析(图2和3B)。首先,每个细胞的平均γ-H2AX荧光通过将测量的每孔(在800nm处)的γ-H2AX荧光除以相应的测量的RedDot-标记的荧光每孔DNA(680nm)(图2)。将每个细胞的γ-H2AX荧光值除以用作媒介物对照的两个孔(在此对应于图1和2中标记为'C'的孔获得的800nm处的平均荧光值)以确定相对于媒介物对照的H2AX水平的磷酸化百分比变化。所获得的数字被定义为遗传毒性指数(或γ-γ2AX倍数诱导),并且在完全健康的细胞中通常为0-2,在用产生colibactin的细胞感染的细胞中为5-7,大肠杆菌MOI 25(图2和图3B)。


图2. γ -H2AX量化的数据分析。 我们如何处理来自原始数据的数据到计算 -H2AX倍数诱导(基因毒性指数)的代表性图像。


图3.用感染的HeLa细胞对来自感染的HeLa细胞的磷酸化的H2AX(γ -H2AX)进行定量分析 + 电子。大肠杆菌或与 pks - 大肠杆菌 :一种。合并检测总DNA(红色,680nm)和γ-em -H2AX(绿色,800nm)的In-Cell Western板图像的实例。在感染E的细胞的高MOI下可以观察到高水平的γ-H2AX。大肠杆菌pks + 。 B.相对于对照(未感染的细胞)计算的γ-γ2AX倍数诱导(与每孔细胞量标准化的γ-H2AX荧光)显示出与由非产生colibactin的细菌感染的细胞相比,取决于产生colibactin的细菌(大肠杆菌pks + +)感染的细胞中的MOI的基因毒性剂量反应(<大肠杆菌菌株 - )。图中表示的数据来自两个生物学重复和两个独立实验。数据使用GraphPad Prism 6.0进行绘图。显示标准偏差(sd)的平均值。

食谱

  1. HeLa细胞培养基
    450毫升DMEM高糖与丙酮酸和GlutaMax
    50毫升胎牛血清
    5毫升NEAA
  2. 固定液(1x PBS,4%PFA)
    5毫升PFA储备液
    5毫升20%10x PBS
    40毫升蒸馏水
    分装并储存在-20°C

    在实验当天解冻一个等份
  3. 中和溶液(20mM NH 4 Cl在PBS中)
    一个。准备在蒸馏水中的储备溶液(1M NH4Cl),并储存在4°C。
    湾将原液溶解在PBS中以达到适当浓度
    C。继续冰,直到使用
  4. 10%Triton X-100

    50μlPBS中的500μlTriton X-100 该解决方案可以在4°C下保存2-3周
  5. 透化溶液(1x PBS,0.2%Triton X-100)
    200μl10%Triton X-100
    10毫升PBS
    继续冰,直到使用
  6. 阻断液(MAXblock阻断介质,1x PHOSTOP抑制剂,RNase [100μg/ ml])

    4.5 ml MAXblock阻挡介质 500μl10x PHOSTOP抑制剂
    50μlRNase储备溶液(PBS中10mg / ml,10mM HEPES pH7.5,15μMNaCl,等分试样储存在-20℃)
    继续冰,直到使用
  7. PST缓冲液(PBS,2%胎牛血清,0.2%Triton X-100)
    48毫升PBS
    1毫升2%胎牛血清

    1ml 0.2%Triton X-100 PBS
  8. 100倍叠氮化物
    将叠氮钠溶于蒸馏水中,终浓度为50 mg / ml(5%w / w),储存于4°C。
  9. 抗-γH2AX溶液(在PST缓冲液中的兔单克隆抗-γ2H2AX[1/200],500μg/ ml叠氮化物)
    1.5毫升PST缓冲液
    7.5μl抗-γ<-H2AX

    15μl100x叠氮化物 注意:此解决方案可重复使用2-3次,并在5°C下保存数周。
  10. 二级抗体溶液(IRDye TM 800CW山羊抗兔[1 / 1,000],RedDot TM 2 [1/1000]在PST缓冲液中)。
    2.5毫升PST
    2.5μlIRDye TM 800CW山羊抗兔
    2.5μlRedDot TM 2 2

致谢

该协议改编自以前发表的作品(Audebert等人,2010; Martin等人,2013; Bossuet-Greif等人, ,2016; Tronnet等人,2017)。这项工作得到了法国国家法律研究所的资助[ANR-13-BSV3-0015-02,ANR-13-BSV1-0028-01]。作者声明没有利益冲突或利益冲突,关于本议定书的发表。

参考

  1. Audebert,M. Riu,A.,Jacques,C.,Hillenweck,A.,Jamin,E. L.,Zalko,D.,Cravedi,J. P.(2010)。 使用γH2AX测定法评估人类细胞系中多环芳烃的基因毒性。 Toxicol Lett 199:182-192。
  2. Bossuet-Greif,N.,Belloy,M.,Boury,M.,Oswald,E。和Nougayrede,J。(2017)。 使用产生colibactin的大肠杆菌菌株感染HeLa细胞的方案和定量诱导的DNA-损坏。 Bio-protocol 7(16):e2520。
  3. Bossuet-Greif,N.,Dubois,D.,Petit,C.,Tronnet,S.,Martin,P.,Bonnet,R.,Oswald,E。和Nougayrede,J.P。(2016)。 大肠埃希菌 ClbS是一种colibactin抗性蛋白。 < em Microbiol 99(5):897-908。
  4. Martin,P.,Marcq,I.,Magistro,G.,Penary,M.,Garcie,C.,Payros,D.,Boury,M.,Olier,M.,Nougayrede,JP,Audebert,M.,Chalut ,C.,Schubert,S.和Oswald,E。(2013)。 大肠杆菌中铁载体和colibactin基因毒素生物合成途径之间的相互作用
  5. Nougayrède,JP,Homburg,S.,Taieb,F.,Boury,M.,Brzuszkiewicz,E.,Gottschalk,G.,Buchrieser,C.,Hacker,J.,Dobrindt,U。和Oswald,E。(2006年)。 大肠杆菌诱导真核细胞中的DNA双链断裂。 科学 313(5788):848-851。
  6. Taieb,F.,Petit,C.,Nougayrede,J.P。和Oswald,E。(2016)。 肠道细菌基因毒素:细胞致密扩张毒素和colibactin EcoSal Plus 7(1)。
  7. Tronnet,S.,Garcie,C.,Brachmann,A.O.,Piel,J.,Oswald,E.和Martin,P.(2017)。 高铁供应抑制致病性大肠杆菌合成基因毒素colibactin 通过非规范的Fur / RyhB介导的途径。 Pathog Dis 75(5)。
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
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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