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FluorescencePlusGiemsa
FluorescencePlusGiemsa

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)

Solutions

10 mM Br-dU

Hoechst 33258

McIlvaine's buffer, pH 7.0

Sorensen's buffer, pH 6.8

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

Dilute 20-fold with water to make 1x SSC


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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