Dulbecco modified eagle medium (dmem)

Human osteosarcoma U2OS and colon cancer HCT116 cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) (Carlo Erba), supplemented with 1% L-glutamine and 10% FBS (fetal bovine serum). U2OS cells were seeded in 35 mm dishes at a concentration of 100000 cell/ml. Filter Units (Millipore), and imidazole was finally removed by buffer exchange with PBS pH 8.0 with Amicon Ultra-15 Centrifugal Filter Units.

The study on amniotic fluid was performed on 25 samples (12 were 46, XY and 13 were 46, XX), established following amniocentesis. A small aliquot of cells was obtained after the analysis and the results of prenatal diagnosis. Written informed consents to use the cells, only for research purposes, were obtained from the patients. All samples were rendered anonymous and stored, at passage 1, with the concentration of 1*10 6 /ml in cryogenic system of our department. After thawing cells were cultured in 100 mm tissue culture Petri (Jet Biofil) at 37 C, 5% CO 2, with DMEM (Carlo Erba) supplemented with 20% FBS 1% glutamine, 1% antibiotics (Gibco).
Copy number variation (CNV) of hTERT was used to evaluate the number of hTERT gene copies to avoid the presence of cryptic rearrangements. We used the following primers: TaqMan Copy Number Reference Assay RNAse P and TaqMan Copy Number Assay hs00233340 (exon 1 TERT) e hs01237576 (exon 16 TERT) (Applied Biosystem).
Data processing was evaluated by software Copy Caller 1.0 (Applied Biosystem).