TRISOMY 8 MOSAICISM: CELL CYCLE KINETICS AND ...

supernatant was added to each well while mixing. The plates were incubated in 100%
humidity incubator at 37 o C, and checked one day after initiation for contamination.
Thereafter, cultures were checked at weekly intervals for clumped cells. The cells were
transferred to a 25cm 2 culture flask when a large number of clumps were present, within
3 to 4 weeks. The samples were subcultured by pouring the contents of the flask into a
15ml tube. The cells were pelleted and the supernatant was removed with a sterile pipet.
Double the amount of fresh media was added to the cell suspensions for a 1:2 split. The
flasks, with the cap slightly loose, were stood on end in the incubator. Subculturing was
performed twice a week.
Chromosomal harvest of lymphoblasts
The cells were subcultured by placing the cell pellet into double the media and
then dividing between two 25cm 2 flasks. The flasks were set on their ends and incubated
at 37 o C for 48 hours in 5% CO2 incubator. Two hours before harvest 0.05ml colcemid
(10ug/ml) was added to each flask. The culture was poured into a 15ml tube and
centrifuged at 1500 rpm in IEC Centra CL2 centrifuge for 7 minutes. All except 1-2ml
media was removed and the cells were resuspended by agitation. Once the cells were
mixed, 0.05ml warm 0.075 M KCl was added. The volume of the tube was brought to
10ml with 0.075 M KCl and mixed by inversion. The culture was incubated at 37 o C for 5
minutes and centrifuged for 7 minutes in IEC Centra CL2 centrifuge. The supernatant
was removed and the cells were resuspended by agitation. A small amount of fixative
was slowly added to the tube and the total volume was brought to 5ml with fixative. The
sample was centrifuged and the supernatant removed. Cells were resuspended with 4ml
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