Cell harvester 8

Cells were seeded in 96-well plates (5×10³ cells/well) and grown during 24 h in complete medium. After 24 h the medium was changed to 2% charcoal-stripped FCS and the cells were treated with the compounds described. The treatment was repeated an additional day with the addition of 0.2 μCi methyl-[³H]-thymidine/well. Cells were harvested in a Nunc Cell Harvester 8 and radioactive nuclei retained into the glass fiber filters were counted in a liquid scintillation counter. The results are expressed as percentage of the control incubated in the absence of any compound. Proliferation response in transfected cells was evaluated by automatic cell-counting (Beckman Coulter Z1 Cell-counter).

Cells were seeded in 12-well plates (15 × 10³ cells/well) for cell-counting or 96-well plates (5 × 10³ cells/well) for DNA synthesis experiments and grown during 24 h in complete medium. After 24 h the medium was changed to 2% charcoal-stripped FBS and the cells were treated with the compounds described. The treatment was repeated an additional day (with the addition of 0.2 μCi methyl-[³H]-thymidine/well for DNA synthesis experiments). Proliferation response was evaluated harvesting cells in Cell Harvester 8 (Nunc, Roskilde, Denmark) and counting in a liquid scintillation counter at 48 h or by cell-counting using Neubauer chamber. The results are expressed as percentage of the control incubated in the absence of any compound.