TRISOMY 8 MOSAICISM: CELL CYCLE KINETICS AND ...

protection from light, which may increase the exchange rate between chromatids.
Cultures were incubated at 37 o C for 72 hours and then harvested in minimal light. The
slides were prepared and then stained with Wright’s stain (Criterion Sciences, Inc.).
Wright’s stain was made in a small test tube by combining 3ml Gurr’s buffer with 1ml
Wright’s stain. A pipette was used to mix and flood the slide with the solution. Two
slides may be stained from one tube of stain. The slides were rinsed with water after 2
minutes and then air-dried. One hundred banded metaphase cells were scored by
microscope location coordinates and identified as being normal or trisomy 8.
Fluorescence Plus Giemsa (FPG) Technique
Sister chromatid exchange slides were destained by placing them in fresh fixative
for 2 minutes. The slides were then air dried and placed in Hoechst 33258 for 15
minutes. Hoechst stain was made by filling a glass coplin jar with Gurr’s buffer and
adding enough Hoescht powder until the solution was pale yellow. The slides were then
rinsed in distilled water and air-dried. The slides were placed in a square petri dish and
flooded with Gurr’s buffer. The dish was placed 5 inches under a 75 watt sun lamp for
25 minutes. The slides were rinsed in distilled water and placed in a new square petri
dish. Several drops of saline sodium citrate (2XSSC) solution, prepared by adding 4ml
20XSSC (175.3g 3M NaCl and 88.2g 0.3M Na3 citrate dissolved in 1000ml distilled H20)
and 36ml distilled H20, were applied to the slide, which was then covered with a glass
coverslip. The covered petri dish was placed in a 65 o C water bath for 15 minutes. The
slides were rinsed in distilled water and air-dried. The slides were stained in 4% Giemsa
(48ml distilled water and 2ml Giemsa stock) for 10 minutes. Finally, the slides were
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