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The Comet Assay


LPI/CCP protocol for the Comet Assay, Alkaline Version
1. Equipment and non-chemical materials  
1.1 Electrophoresis power supply: Thermo EC 1000-90 or alternative
1.2 Electrophoresis tank: Large Fisher Scientific Recirculating Horizontal System or alternative
1.3 Frosted slides: Surgipath or alternative
1.4 Coverglasses: Surgipath 24 x 50 mm or alternative
1.5 Microcentrifuge and microcentrifuge tubes
1.6 Various micro-pipet and pipet tips
1.7 Disposable test tubes
1.8 Dishes for lysis of cells: Wheaton 900170
1.9 Nikon E400 Fluorescence microscope or equivalent
1.10 Refrigerator and other general laboratory items, e.g. balance and pH meter
 
2. Chemicals and reagents
2.1 10 x concentrated Dulbecco's PBS, Mg2+ and Ca2+ free
    NaCl (Sigma S7653) 80 g    
    KCl (Fisher Scientific P217-500) 2 g    
    Na2HPO4.2H2O (Fisher Scientific S472-500) 11.5 g    
    KH2PO4 (Sigma P0662) 2 g    
  Adjust pH to 7.2 and q.s. to 1 liter with dd H2O, autoclave store at RT.
   
2.2 Dulbecco's PBS, Mg2+ and Ca2+ free
  Dilute 10 times of solution 3.1 with dd H2O, final pH 7.3 ~ 7.4. Autoclave at 121C for 20 min, store at RT.
   
2.3 Lysing solution for the comet assay (equivalent to 2 liters in the final preparation with the addition of 1% Triton X-100 and 10% DMSO)
  2.4 g Tris (Sigma T6066), 74.4 g Na2EDTA.2H2O (Sigma E5134), 292.2 g NaCl (Sigma S7653), appropriate amount of H2O.
  Set pH to 10 with NaOH pellets (Sigma S0899), q.s. to 1780 ml with dd H2O, store at RT.
   
2.4 10 N NaOH (Sigma S0899) stock solution (Caustic!!! Handle with care)
  200 g q. s. to 500 ml with dd H2O, store at RT.
   
2.5 200 mM EDTA (Sigma E5134) stock solution
  Dissolve 74.4 g in dd H2O, set pH to 10 with NaOH, q. s. to 1000 ml with dd H2O, store at RT.
   
2.6 0.5% low melting point agarose (LMPA)
  0.5 g + 100 ml PBS(A), boil in microwave oven to dissolve agarose, compensate the evaporated H2O, aliquot the agarose into 15 ml tubes (~3 ml/ tube) before gelation and store the tubes under 4C.
 
2.7 1.0% normal melting point agarose (NMPA)
  1 g + 100 ml dd H2O, microwave to dissolve agarose, compensate the evaporated H2O, cool down to around 60C before use.
 
2.8 Neutralizing buffer - 0.4 M Tris (Sigma T6066)
  Dissolve 48.5 g in dd H2O, set pH 7.4 with HCl and q. s. to 1000 ml with dd H2O, store at RT.
   
2.9 Electrophoresis solution 300 mM NaOH, 1 mM EDTA
  Mix 48 ml of 10 N NaOH, 8 ml of 200 mM EDTA and 1544 ml dd H2O. Chill to 4C before use.
   
2.10 Fluorescent staining solution 250 x Strength Stock
Ethidium bromide (Sigma E8751, DNA poison!!! Handle with care) in dd H2O 5 mg/ml, store at RT.
   
2.11 Staining solution
  Dilute solution 3.10 250 time to 20 g/ml with dd H2O, store at RT.
   
2.12 Dimethyl sulfoxide (Fisher Scientific D128-500)
   
2.13 Triton X-100 (Sigma T9284)
   
3. Operation procedures
3.1 Material and reagent preparation
   
3.1.1 Mix 89 ml lysing solution (3.3) with 1 ml of Triton X-100 and 10 ml of DMSO in a staining dish, chill the mix to 4C before use.
   
3.1.2 Microwave the LMPA and NMPA aliquots to complete molten state (in a container filled with water), check and shake the tube every a few seconds to avoid overheat and explosion. Leave the molten LMPA in 40C bath and NMPA in 45C for at least 10 min before use.
   
3.2 Slide preparation
   
Note: Steps 3.2.1 may be carried out days before Comet assay experiment.
   
3.2.1 Dip of clean frosted slides (unfrosted portion), one at a time, in molten NMPA. Scrape agarose off the underside. Leave the slides horizontally to dry. A slide warmer may be used to facilitate the drying of slides.
   
Note: Conduct the steps 3.2.2 to 3.2.3 under dimmed light to prevent the occurrence of DNA damage.
   
3.2.2 Mix about 10,000 cells with 75 l of molten 0.5% LMA at 37C. Pipette the cell suspension onto the first agarose layer, and spread the mixture with a coverglass, and maintain the slide on an ice-cold flat tray for 30 min.
   
3.2.3 After removal of the coverglass, add the third layer of 0.5% LMA (70 l) at 37C, spread gel using a coverglass and again allow the gel to solidify on ice for 10 min.
   
3.2.4 After removal of the coverglass, immerse the slide in freshly prepared cold lysing solution for a minimum of 1 h at 4C.
   
3.3 Electrophoresis
   
3.3.1 Remove the slides from the lysing solution, drain the slides and rinse briefly with cold dd H2O. Place in a horizontal gel electrophoresis tank side by side, avoiding spaces and nearest to the anode. Fill the electrophoresis tank with 1.6 L of cold electrophoresis solution.
   
3.3.2 Leave the slides in the solution for 30 min to allow the unwinding of the DNA and expression of alkali-labile damage.
   
3.3.3 Perform electrophoresis at 4C for 30 min (or other desirable time) at a constant voltage (0.8 V/cm).
   
3.4 Staining
   
3.4.1 After electrophoresis, Place the slides horizontally and gently add Tris buffer (0.4 M Tris, pH 7.5) dropwise to neutralize the excess alkali. Let the slides sit for 5 min. This neutralizing procedure was repeated three times.
   
3.4.2 Add 60 l EtBr (20 g/ml) to each slide and cover it with a coverglass. Let the slides sit for 5 min at the horizontal position. Place the slides in a humidified airtight container to prevent drying of the gel. Analyze the slides within 3-4 h.
   
3.5 Slide scoring
Randomly score the comet-like nuclei using the Comet Assay Image Analysis System. For each treatment score a minimum of 50 cells or 25 cells per slide is required.