Figure 2. Suppression of pulp cell proliferation by NO. (A) The pulp removed from normal one-week-old mice was cultured for 1 wk to allow for the outgrowth of pulp cells, which were plated onto normal plastic dishes or type IA collagen gel-coated dishes and cultured in MEM + 20% FCS in the absence or presence of 50 µM NOC-18. The cells were observed under a phase-contrast microscope at 14-day culture. Note that the pulp cells on the collagen-coated dishes grew faster than those on the uncoated dishes. (B) The number of viable cells was counted at various time-points by trypan-blue dye exclusion assay after culture on collagen-coated dishes in the absence (circle) or presence of 50 µM NOC-18 (square). *The density of cells exposed to NOC-18 was significantly lower than that of cells cultured for the same period without NOC-18 (n = 4). (C) Concentration-dependent suppression of cell growth by NOC-18. *Significantly lower than the control without NOC-18 (n = 4). (D) Incorporation of BrdU (left panels) by pulp cells cultured for 5 days in the absence (upper panels) or presence of 50 µM NOC-18 (lower panels). All nuclei in the same fields were visualized with Hoechst 33258 (right panels). (E) The BrdU incorporation at day 5 was analyzed by a flow-cytometer. The upper and lower panels show the incorporation of BrdU by the control and NOC-18-treated cells, respectively. After incubation with BrdU, the cells were treated with (thick lines) or without (thin lines) the antibody for BrdU. The ratios of BrdU-incorporated cells were 50.63% for the control cells and 32.42% for the NOC-18-treated cells.