Sister Chromatid Exchange (SCE) Stain
Materials
1. Bromodeoxyuridine (BrdU) stock solution, 1 mg/ml in PBS, HBSS, or SF-DMEM.
2. Hoechst 33528 (bisbenzimide) stock solution, 10 mg/ml
3. McIlvaines buffer pH8
Sol.A-0.1 M Citric Acid
2.25 ml
Sol.B-0.2 M Disodium phosphate
97.75 ml
4. 6% Geimsa in Gurr's Buffer (Gibco/BRL) pH6.8
Procedure
1. Add BrdU to growing cultures to a final concentration of 10 ug/ml. Incubate for 2 cell
cycles, 36 to 60 hours, depending on the cell line.
2. Harvest cells for metaphase chromosomes as usual. Do not dry the slides on a slide
warmer or heat.
3. Stain slides in Hoechst 33528 (200 ug/ml) in water for 30 min. At room temp. Rinse
well with water and air dry or blot dry, do not heat.
4. Place 100 ul of McIlvaines buffer on the slide and cover with a 22x60 mm coverslip.
5. Place slide on slide warmer at 55-60C under long and short wave UV light for 20 min
(we use a UV crosslinker). Rinse slide with water and blot or air dry.
6. Stain in Geimsa for about 10 min.
From: Goto, K., S. Maeda, Y. Kano, T. Sugiyama. Factors involved in differential
geimsa-staining of sister chromatids. Chromasoma. 66:351-359, 1978.