C26 tumor proliferation. a) Tumor mass kinetics, measured upon tumor explant at the indicated times. After a lag time of about two weeks, tumor growth is linear and results in a mass of considerable size (>2 g, i.e. about 10% of the body weight). The mean ± SEM of at least three independent experiments performed in triplicate is shown (for each data point, the number of replicates, "n", is: 9 < n < 22). b) The DNA profile by flow cytometry of PI-labeled cells, at 3 weeks following transplantation, shows: a significant sub-population of cells in the S phase (M3), which indicates that tumor cells are actively proliferating; a haploid, sub-G1 peak (M1) that does not resemble apoptotic cells/debris; lack of polyploidism; M2 and M4 indicate the G1 and G2 sub-populations, respectively. c) BrdU incorporation (red) was detected in the tumor cells at 3 weeks following transplantation, the day after IP injections of two doses (50 mg/Kg of body weight) of BrdU 4 h apart (+ BrdU) though not in the sham-injected mice (-). Nuclei are counterstained with Hoechst (blue). Bar = 100 μm. Inset, confocal phase contrast image merged with the red fluorescence reveals the presence of BrdU+ and negative nuclei (black and white arrow, respectively). Bar = 10 μm d) Flow cytometric analysis of BrdU incorporation in cells enzymatically extracted from C26 tumors at 3 weeks following transplantation. BrdU+ cells were plotted against PI labeling (bottom) and compared to C26 cells from sham-injected mice. Cells above background are in R2 and R3.