Cells detached with trypsin/EDTA as for routine subculture were plated in 8-well chamber slides (Nunc, Rochester, NY) at 40,000 cells per well in 0.2 mL growth medium. On the second day the growth medium was removed, the wells washed with serum-free medium and the incubation continued with 0.2 mL serum-free DMEM containing 1 mg/mL crystalline BSA (DMEM-BSA). On the third day the DMEM-BSA was removed and replaced with 0.2 mL DMEM-BSA containing the experimental additives including human IGF-I (Gropep, Inc., Adelaide, Australia) human IGF-II (Gropep, Inc.) and human PDGF-AB (Upstate Biotechnology, Inc., Lake Placid, NY). After 24 hours the medium was removed and the slides were fixed with 2% paraformaldehyde in phosphate buffer (0.1 M Na2 HPO4, pH 7.0) for 1 hour at room temperature, washed with PBS (0.01 M Na2HPO4, 0.15 M NaCl, pH 7.4), permeabilized by a 10-minute treatment with PBS containing 0.1% Triton X-100 and blocked with 20% nonimmune goat serum (The Binding Site, Ltd., Birmingham, UK) in PBS for 60 minutes at room temperature. Primary and secondary antibody treatments with anti-Ki67 (Dako North America Inc., Carpenteria, CA) and Cy2-conjugated goat anti-mouse antibody (Jackson Immunoresearch Inc., West Grove, PA) were for 60 minutes at room temperature using 2% goat serum in PBS with three 5-minute washes in between. The slides were then rinsed briefly with PBS, reacted with 1 μg/mL 4′, 6-diamidino-2-phenylindole (DAPI) in PBS for 5 minutes and were rinsed again with PBS. Photomicrographs were taken with a microscope equipped with epifluorescence illumination and phase-contrast optics (Optiphot; Nikon, Tokyo, Japan) and with a digital camera (RETIGA EXi; QImaging Corp., Burnaby, Canada). Successive paired photomicrographs of Ki67-associated and DAPI fluorescence were taken during a north to south transit across the center of each well were quantified by an observer naïve to the experimental conditions counting a minimum of three successive fields and a minimum of 300 DAPI-positive cells. Images were assembled into composite photographs with image management software (Adobe Photoshop C2; Adobe Systems, Inc., San Jose, CA).