TRISOMY 8 MOSAICISM: CELL CYCLE KINETICS AND ...

fixative, and the centrifugation and resuspension with 4ml fixative was repeated. Finally,
the sample was centrifuged and all except 0.5 to 1ml of supernatant was removed,
depending on the pellet size. The cells were mixed by gently pipetting. The cells were
prepared for the slide making procedure.
Fibroblast Cultures
The placental tissues were placed on the top half of a sterile 100mm petri dish
containing Amniomax culture medium. The amnion, chorion, and villi were minced into
small pieces, while the blood vessel (fetal tissue) was removed from the umbilical cord
and minced. The pieces were placed into five 35mm culture plates and incubated for 4
hours at 37 o C. Culture media (1ml) was carefully added so as not to dislodge the
explants. Cultures were checked every 48 hours and the media was changed at least once
a week by aspirating off the old media and adding 2ml Amniomax at room temperature.
The cells grew from the explant, usually first as epithelial (round) cells and then as
fibroblasts (elongated). To subculture the cells, 2 to 3 dishes with good fibroblast growth
were selected. The media was removed and the dish was rinsed with HBSS, without
Mg +2 and Ca +2 . To each dish, 0.5ml or less of trypsin/EDTA was added and the sample
was then placed in the 5% CO2 incubator at 37 o C for 5 minutes. The flasks were scanned
under an inverted microscope for the loosening of cells; tapping the bottom of the plate
helped to dislodge the cells. When the cells were detached, 2ml of media were added to
stop the trypsin protease action. A new flask was prepared to receive 1.5 ml of cells, as
0.5ml was replaced into the original dish. The flasks were returned into the incubator.
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