Bleomycin Sulfate

ADIPO-P2 cells are grown in D-MEM high glucose medium supplemented with 20% fetal calf serum, penicillin (100 U/mL) and streptomycin (100 μg/mL) at 37 °C and 5% CO₂ atmosphere. Cells are cultured as monolayer in TC25 Corning flasks containing 1.5 × 10⁵ cells/mL. For each experiment, two flasks are set up, one for the control and one for the treated culture. During the log phase of growth ADIPO-P2 cells are treated with a 30 minutes pulse of 2.5 μg/mL of Bleomycin sulfate. Control cultures are set up in parallel but not exposed to Bleomycin sulfate. Time of exposure and concentration of Bleomycin sulfate are chosen according to previous studies carried out in our laboratory with mammalian cells exposed to Bleomycin sulfate. At the end of the pulse treatment with Bleomycin sulfate, the cells are washed twice with Hank's balanced salt solution and kept in culture with fresh culture medium until harvesting. Cells are continuously maintained in culture during 5 passages or subcultures after treatment. Subcultivation is carried out whenever the cultures became confluent (approximately 4 × 10⁵ cells/mL of culture medium). To estimate cell growth, at the time of subcultivation cells are collected by trypsinization, an aliquot of about 200 μL stained with 0.4% trypan blue, and the number of viable cells is determined. Cells are then suspended in fresh culture medium and dispensed into new culture flasks containing 1 × 10⁵ cells/mL to continue growing. The rest of the cells is discarded or dispensed in another flask for cytogenetic analysis, which is performed at 18 hours and 10 days after the end of treatments. To analyze chromosomal aberrations, colchicine (0.1 μg/mL) is added to cell cultures during the last 3 hours of culture. Chromosome preparations are made following standard procedures. After harvesting, cells are hypotonically shocked, fixed in methanol:acetic acid (3:1), spread onto glass slides and processed for PNA-FISH. Two independent experiments are carried out.