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The Micronucleus Assay


LPI/CCP protocol for in vitro micronucleus assay using CHO-K1 cells
1. Equipment and materials
1.1 Tissue culture vessels (e.g. flasks or plates)
1.2 CHO-K1 cell line (from ATCC)
1.3 Rat liver S9 (Aroclor-1254 induced), if metabolic activation system is to be used
1.4 9 disposable Pasteur pipets
1.5 Frosted slides (Surgipath #00240 or alternative)
1.6 Coverglasses (Surgipath 24 x 50 mm, #00145, or alternative)
1.7 General purpose bench-top centrifuge (Fisher Scientific Marathon 3000)
1.8 Conical centrifuge tubes (Falcon, VWR #21008-918)
1.9 Wheaton stainless steel 30-slide racks and glass staining dishes
1.10 Various micro-pipets and pipet tips
1.11 Nikon E400 fluorescence microscope or equivalent
1.12 Other general laboratory items, e.g. freezer, balance and pH meter
 
2. Chemicals, reagents and media
2.1 Ham's F12K medium (Mediatech, through Fisher Scientific) supplemented with 2 mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate (Fisher Scientific MT25035CI), 10% fetal bovine serum (FBS)
     
2.2 Ham's F12K medium supplemented with 2 mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate
   
2.3 Trypsin-EDTA solution, (Fisher Scientific ICN1689649)
   
2.4 Cytochalasin B (VWR IC19511910,), 3 mg/ml in DMSO, sterile
   
2.5 Dulbecco's PBS (A), Mg2+ and Ca2+ free (may be prepared from 10 x stock solution)
  Ingredient per 1000 ml
 
NaCl (Sigma S7653) 8.0 g    
    KCl (Fisher Scientific P217-500) 0.2 g    
    Na2HPO4 (Fisher Scientific S472-500) 1.15 g    
    KH2PO4 (Sigma P0662) 0.2 g    
  Adjust pH to 7.3 ~ 7.4 and q.s. to 1 liter with dd H2O. Autoclave at 121C for 20 min. Keep sterile and store at RT.
   
2.6 Fixative, made fresh
  Ingredient per 26 ml  
    Methanol (Fisher Scientific A452-4) 25 ml    
    Acetic acid, glacial (Fisher Scientific A35-500) 1 ml    
   
2.7 10 g/ml acridine orange (VWR # EM-1125) in PBS (A), good for 1 week on bench
   
2.8 Potassium/magnesium solution (for S9 mix)
  Ingredient per 500 ml  
    KCl (Fisher Scientific P217-500) 61.5 g    
    MgCl2 6H2O (Fisher Scientific, BP214-500) 40.7 g    
    dd H2O to 500 ml    
  Dissolve the salts. Autoclave at 121C for 20 min. When the solution has cooled slighted, tighten cap. Keep sterile and store at RT.
   
2.9 0.2 M Sodium phosphate buffer, pH 7.4 (for S9 mix)
    Ingredient per 500 ml  
    NaH2PO4 (Fisher Scientific BP329-1) 12.0 g    
    Na2HPO4.2H2O (Fisher Scientific, S472-500) 17.8 g    
    dd H2O to 500 ml    
  Dissolve the salts with 450 ml dd H2O. Adjust pH with 0.2 M NaH2PO4 or 0.2 M NaHPO4 to 7.4. Add dd H2O to the final volume of 500 ml.
Autoclave at 121C for 20 min. When the solution has cooled slighted, tighten cap. Keep sterile and store at RT or 4C.
   
2.10 80 mM NADP (VWR# 80053-340, for S9 mix)
  Ingredient per 1 ml
    NADP (F.W. 765.39) ~61.23 mg  
    Sterile dd H2O ~1 ml*  
  It is better to weigh a batch of dry aliquots of NADP in small sterile tubes with tight caps. Avoid light and moisture. Store them at -20C or below. Add dd H2O according to the exact weight of NADP upon use. This is an expensive reagent make up solutions as required and not in excess.
   
2.11 120 mM D-Glucose-6-phosphate (G-6-P, VWR ICN100312, for S9 mix)
 
Ingredient per 1 ml    
    G-6-P (F.W. 304) ~36.48 mg    
    Sterile dd H2O ~1 ml*    
  Prepare as for NADP above.
   
2.12 Fresh High S-9 mix with cofactors
    Ingredient per 10 ml final concentration    
    Sterile dd H2O 2.8 ml      
    0.2 M phosphate buffer, pH 7.4 5.0 ml 0.1 M    
    80 mM NADP 0.5 ml 4 mM    
    120 mM G-6-P 0.5 ml 6 mM    
    K, Mg salts solution 0.2 ml 33 mM 8 mM    
    Rat liver S-9 (Aroclor-1254 induced) 1.0 ml 10% v/v    
  Actual volume should be determined by each experiment. Make up solutions as required and not in excess. Prepare immediately before use in ice bucket in above order. Keep the ingredients chilled in preparation. Add in S9 fraction last. Discard any leftover for S9 or S9 mix. Never re-use S9 or S9 mix.
   
3. Operation procedures
3.1 Culture CHO-K1 cells in ATCC recommended growth medium in exponentially proliferating status. Prepare a cell suspension at a density ~1 106 cells /ml. Seed cells in culture vessels at 3 104 cells /0.3 ml (cell suspension : medium = 1 : 9)/cm2. The number of subcultures is determined by the following formula:
(# of dose levels) x (# of treatment regimen, i.e. 4 h/20 h, +S9/-S9) x (3 replicates)
3.2 Grow the subcultures until they reach 60 ~ 70% confluence.
3.3 Prepare the treatment medium by adding the test compound to a desirable amount of growth medium (including 3 g/ml cytochalasin B, for 20 hr treatment) or serum free medium (for 4 hr treatment). Use metabolic activation system (rat liver S9) for 4 h treatment when necessary. Normally negative and positive controls should be included.
3.4 Remove the medium in the culture vessels by aspiration and immediately add the same amount of treatment medium in the vessels. Do not expose the cells to air for more than a few seconds.
3.5 Skip this step for 20 hr treatment experiment. For 4 h treatment, remove the treatment medium by aspiration. Wash the cells with a same amount of serum free medium once. Remove the serum free medium and add fresh growth medium, including 3 g/ml cytochalasin B. Culture the cells for another 20 h.
3.6 Remove the growth medium from cultures by aspiration. Wash the monolayer twice with the same amount of PBS (A). Remove PBS (A) and add 40 l/cm2 of Trypsin-EDTA to the culture vessel. Incubate and check from time to time the cells. When all the cells are detached, add 0.3 ml/cm2 of growth medium to stop trypsinization.
3.7 Pellet the cells at 250 x g, 5 min in conical centrifuge tubes. Aspirate most of the medium, only leave about 0.5 ml.
3.8 Resuspend the cells in single cell suspension by vigorous pipeting or vortex mixing. Set vortex mixer to a low/intermediate speed. Place a cell suspension-containing tube on the top and slowly add 5 ml of cool fresh fixative. Leave the tubes on bench for 15 min.
3.9 Pellet the cells at 250 x g, 5 min. Decant the fixative to an organic waste bottle. Add fresh fixative (~100,000 cells/ml) to the tube.
3.10 Resuspend the cells using a 9 Pasteur pipette or using intermediate vortex mixing. Drop 8-10 drops of the cell suspension on a horizontally held slide. For each independent treatment, at least 3 slides should be made. Leave the slides on a bench and air-dry the slides. Check the cell density with a phase contrast microscope (40x Ph objective, condenser at Ph2).
3.11 If cells are not to be scored immediately, the dried slides may be stored in N2 filled zip bag in 20C until use.
3.12 After thoroughly air dry, stain the slides with 10 g/ml acridine orange for 2 min. Rinse the slides with dd H2O for 4 min. Immediately cover the wet slides with coverglasses. Keep the cells on slide moisturized during the scoring. Score the slides under a 20x objective. Use higher magnification to examine the details.
3.13 Following micronucleus scoring criterion, score 1000 binucleated cells per independent culture.