Figure 3. Growth curves of primary REF cells and rapamycin-derived cell lines. (A) Number of cells as a function of time (days). REF cells passage 3, rapamycin-derived cells (Rapa) passages 10 and 30 were seeded and counted every 24 h for 5 d. The population doubling time (PDT) Td was calculated by using formula Td = (log2)*t/[log2(Nt/N0)], where t is time in hours, Nt is the cell number at time t, and N0 is the cell number at the initial time. Each experiment was performed in triplicate. (B) Cloning efficiency of primary REF cells and rapamycin-derived cells. Ctrl, primary REF cells (passage 3); Rapa, rapamycin-derived cells (passages 10 and 30) were plated at clonal density (100 cells/60-mm plate) and left to grow for 14 d, when they formed eye-visible cell clones. Cells were stained by Giemsa dye to see and count visible clones in light microscope, magnification 10 × 40.