Figure 4. Tracking cell proliferation of CD4⁺ T cells post-treatment with rapamycin. T-Rapa and control cells were transduced with GFP-LV on day 3 of cell culture. (A) Cells were labeled with cell proliferation dye e670 (CPe670) on day 4, day after LV infection, and evaluated by flow cytometry on day 6, 7 and 8 of cell culture. Flow cytometry data represents the CPe670 dilution (black line; modeled populations of proliferating cells in red line) in GFP-LV transduced and NT T-Rapa and control cells on day 6. The number of cell division is shown in the top of each panel. (B) Percentage of cells divided was calculated using FlowJo software. (*p < 0.0174; **p < 0.0017). (C) At day 8 of cell proliferation analysis, the cells were also stained with annexin V. (GFP: *p < 0.0122; NT: *p < 0.0401). (D) At day 6, cells were harvested, counted and replated at 0.5 × 10⁶ cells/ml. NT-CD4⁺ T cells were also evaluated. Cells were maintained under CD3 and CD28 costimulation in media containing cytokines and enumerated every 3 d. The data represent the mean (± SEM) of three independent experiments.