TRISOMY 8 MOSAICISM: CELL CYCLE KINETICS AND ...

insed with water and allowed to air dry. Metaphase cells previously located were
evaluated for number of cell divisions. One division was noted when the chromatids were
equally stained. Two divisions were determined by half of the chromatids stained dark
and the other half stained pale. Lastly, three divisions were designated by one-fourth of
the chromatids stained dark and three-fourths of the chromatids stained pale.
Lymphoblast Transformation
Cord blood was transformed with Epstein Barr virus, which stimulated B-cell
proliferation. In a 50ml sterile centrifuge tube containing 5ml–10ml of cord blood, an
equal amount of RPMI 1640, containing 1% antibiotics (10,000units/ml Penicillin and
sodium and 10,000µg/ml Streptomycin sulfate in 0.85% saline), was added to the tube
and mixed by inversion. A 15ml centrifuge tube was prepared with 3ml Histopaque-1077.
The blood mixture(8ml) was carefully overlaid into the Histopaque tube with a pipette.
The tube was centrifuged at 400 g for 35 minutes at room temperature. Mononuclear
cells were in the luminescent band between the serum/media top layer and the
Histopaque bottom layer. The mononuclear cell band was carefully removed and placed
in a clean 15ml centrifuge tube. The volume of the tube was brought up to 15ml with
RPMI 1640 and mixed by inversion. The sample was centrifuged at 400 g for 20
minutes. The supernatant was removed and the cells were resuspended by vortexing.
The tube was filled with 15ml RPMI 1640 medium and mixed well. The sample was
centrifuged for 20 minutes. The supernatant was removed and the cells were resuspended
by vortexing in 10ml RPMI 1640, containing 1% antibiotics, and 15% heat-inactivated
fetal bovine serum. Next, 40ul of 5mg/ml cyclosporin was added. The sample was
distributed into several wells of a 12 well plate. Next, 0.5ml Epstein-Barr virus filtered
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