Fluorescence Plus Giemsa (FPG) staining of metaphase chromosome spreads

This procedure will enable easy visualization of sister chromatid exchanges (SCEs) in metaphase spreads with a light microscope. Cells are grown in Br-dU (5-bromo-deoxyuridine) for two complete cell cycles. A metaphase chromosome pair with no exchanges will have one sister with both strands labeled by Br-dU and the other sister with only one of two strands labeled. The chromatid with Br-dU in both strands resists the stain and appears light, while the other chromatid with Br-dU in only one strand stains better and appears dark. If an SCE has occured in the first cell division, the sisters can be seen to have traded light- and dark-staining segments. SCEs in the second division are not differentially stained by this procedure. The observed number of SCEs is thus the number per cell division.

1. Split cells and plate cells at a confluence of between 5% and 10%. Let grow unperturbed for 24 hours to fully recover from the splitting procedure.
   
2. Add Br-dU to a final concentration of 20 μM and allow cells to grow in the dark for two cell divisions.
   
   • 20 μM Br-dU is 6.14 μg/mL. Add ¹/₅₀₀th volume of a 10 mM stock solution
     
   • it is important that cells be grown no more and no less than two cell divisions. take care to measure the doubling time of each cell line you will be using since these can vary widely between lines
     
   • cover plates with a layer of aluminum foil to prevent light exposure. light can induce SCEs in cells labeled with Br-dU
     
3. make metaphase ChromosomeSpreads on microscope slides
   
4. immerse slides in 10 μg/ml Hoechst 33258 in water for 20 minutes or try the alternative staining protocol listed below
   
   • dilute ¹/₁₀₀th volume of a 1 mg/ml stock of Hoechst 33258 in water
     
5. rinse slides by dipping in Sorensen's buffer (pH 6.8), add a few drops of Sorensen's buffer (pH 6.8) and add a coverslip
   
6. expose slides on a 55C heatblock to long wavelength UV light (approximately 365 nm) for 20-30 minutes with the coverslip on so the slides don't dry out
   
   • for example, at a distance of 5-10 cm from two 20 watt BLB (black light blue) bulbs
     
7. remove and discard the coverslip
   
8. incubate slides for 1-hour in 1X SSC at 50C.
   
9. stain slides in 10% GiemsaStain in Sorensen's buffer for 20 minutes
   
   • dilute giemsa stock solution by adding 19 volumes of water to yield a 5% staining solution
     
10. rinse briefly by dipping slides in distilled water and let dry
    
11. observe SCE's using brightfield microscopy
    
    • use a 63x or 100x high-quality oil-immersion objective lens
      

Alternative staining protocol

1. treat cells with BrdU and make metaphase spreads as directed above
   
2. stain in 0.1 mg/ml acridine orange for 5 minutes at room temperature
   
3. rinse with running water for 2 minutes
   
4. incubate 1 minute in Sorensen buffer
   
5. add a coverslip and observe by fluorescent microscopy using a FITC filter set (blue excitation, green emission)
   

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Solutions

10 mM Br-dU

• dissolve 100 mg Br-dU powder in 32.6 mL PBS. stable in the dark at 4C for up to 6 months.
  
  • Br-dU is very light sensitive (photodegrades via free-radical formation). do not expose to light
    
• Br-dU stock solution can also be aliquoted and stored indefinitely at -80C. do not refreeze aliquots -- leave at 4C in the dark once thawed
  

Hoechst 33258

• dissolve Hoechst 33258 in water to make a 1 mg/ml solution.
  
  • do NOT use a phosphate buffer to make the concentrated stock solution as Hoechst 33258 is insoluble at high concentration in the presence of phosphate
    
• store at 4C in the dark for up to 6 months
  

McIlvaine's buffer, pH 7.0

• mix 194.5 ml of 0.2 M Na₂HPO₄ with 5.5 ml of 0.1 M citric acid
  
• pH 7.0 with HCl
  
• store at room temperature
  

Sorensen's buffer, pH 6.8

• mix equal volumes 0.1 M Na₂HPO₄ and 0.1 M KH₂PO₄
  
• store at room temperature
  

20x SSC
Final 20x concentration is 3 M for NaCl and 300 mM for sodium citrate.

• Dissolve 175.3 g of NaCl and 88.2 g of sodium citrate dihydrate in 800 ml H₂O.
  
• Adjust to pH 7.0 with HCl if necessary (but it won't in fact be necessary).
  
• Adjust volume to 1 liter.
  

Dilute 20-fold with water to make 1x SSC

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References

Wolff and Afzal, PNAS 93, p5765-5769 (1996).
Perry and Wolff, Nature 251, p156-158 (1974).
Yankiwski, Noonan and Neff, BMC Cell Biology 2:11 (2001).

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Comments

• The fluorescence plus giemsa staining procedure gives permanently stained spreads that can be observed under a light microscope. The alternative protocol with acridine orange gives spreads that need to be observed using a fluorescent microscope and that are not permanent as the acridine orange will eventually (potentially quite rapidly) be photobleached by light excitation.
  
• A microscope with good optics is absolutely required. At a minimum, a 63x plan apochromatic oil-immersion objective lens with a numerical aperture of 1.4 is necessary. Otherwise, the differentially stained sister chromatids will fail to resolve, and the lighter staining chromatid will appear washed out to the point of invisibility. this assay is impossible with inferior quality optics.