TRISOMY 8 MOSAICISM: CELL CYCLE KINETICS AND ...

Peripheral and Cord Blood preparation
METHODS AND MATERIALS
Chromosomal media was prepared by mixing 100ml RPMI 1640, 20ml Fetal
Bovine Serum, 1.3ml phytohemagglutinin (PHA) (Gibco 10576-015), 1.3ml Penicillin
and Streptomycin (10,000 units/ml Penicillin and sodium and 10,000 µg/ml Streptomycin
sulfate in 0.85% saline), and 1.3ml L-glutamine (29.2 mg/ml in 0.85% NaCl). Media
tubes were pre-prepared in 15ml centrifuge tubes with 9ml aliquots of media and then
frozen until needed. The tube of media was removed and thawed and 0.5ml–1ml of whole
blood was added. The blood was incubated in chromosomal media, with PHA, to
stimulate the proliferation of T-cell lymphocytes. The sample was incubated at 37 o C for
72 hours. Colcemid (10µg/ml) was added at 80µl to each sample and incubated at 37 o C
for 20 minutes. Colcemid, which disrupts spindle fiber formation, was added to prevent
the cells from dividing and therefore suspending them in metaphase for chromosomal
analysis. The sample was centrifuged in an International Equipment Company (IEC)
Centra CL2 centrifuge at 1200 rpm for 8 minutes, followed by aspiration and
resuspension in hypotonic medium. The hypotonic solution consisted of 10ml 0.075M
KCl at 37 o C. The tube was incubated in a 37 o C water bath for 9 minutes. The sample
was preserved by adding 2ml cold fixative (3 parts cold anhydrous methanol and 1 part
glacial acetic acid) and mixed by inverting the tube. The sample was centrifuged in the
IEC Centra CL2 centrifuge at 1200 rpm for 8 minutes. The supernatant was aspirated and
then 10ml room temperature fixative was added to the tube. The sample was allowed to
stand at room temperature for 20 minutes, followed by centrifugation for 8 minutes. The
supernatant was removed and 10ml room temperature fixative was added to the tube. The
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