TRISOMY 8 MOSAICISM: CELL CYCLE KINETICS AND ...

Chromosomal Harvest of Fibroblast Cultures
The newly subcultured flask was checked under the inverted microscope for
proper cell confluence. Two to three flasks, with rounded cells, were selected and 0.1ml
of 10mg/ml colcemid was added, gently rotating the flask to mix. The culture was
incubated at 37 o C for 3 to 4 hours. All of the media was pipetted off and placed in a 15ml
tube. The flasks were rinsed with HBSS without Mg +2 and Ca +2 . The supernatant was
removed and added to the centrifuge tubes. Each flask was filled with 0.5 to 1ml trypsin
EDTA and incubated for 5 minutes. When the cells became detached 2ml HBSS with
Mg and Ca was added to stop the trypsin action and the cell suspension was placed in a
centrifuge tube. The sample was centrifuged at 1000-1200 rpm in IEC Centra CL2
centrifuge for 5 minutes and the supernatant was discarded without disturbing the pellet.
Slowly 10-12ml room temperature 0.075 M KCl was added while mixing the cells. The
sample was placed in the 37 o C incubator for 8 minutes. The cells were centrifuged for 5
minutes and the supernatant was discarded. To the tube, 5ml fresh cold fixative was
added very slowly while mixed into a fine suspension. The suspension was refrigerated
for 1-2 hours. The sample was centrifuged for 5 minutes, the supernatant was discarded,
and the cells were resuspended with new fixative. The sample was centrifuged for 5
minutes and all except 0.5-1ml fixative was removed. The cells were gently resuspended
with a pipette. The cell solution was prepared for slide making procedure. Once made,
the fibroblast slides were dried overnight at 50-55 o C.
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