CFSE staining demonstrates multiple rounds of proliferation in both the lymphocyte and APC populations of the cocultivated cells.

(A) Three days after CFSE staining, the lymphoblast population was identified by flow cytometry scatter parameters. The lymphoblast population was biphasic with the majority of cells undergoing slower replication and a subset of cells that demonstrated a higher rate of proliferation. To determine the number of divisions, the fluorescence histogram was overlaid on a grid so that the center of each peak was equidistant from the next peak and represented one cycle of cell division. The number of divisions was determined by counting the number of peaks. (B) The change in fluorescence intensity was measured by determining the distance between the initial peak on day 3 and the shift in fluorescent intensity of equidistant peaks observed on day 5. The fluorescence shift indicates that 8 rounds of cell division have occurred by day 5. (C) The APC population was identified by flow cytometry light scatter parameters and the replication rate determined as described in panel A. (D) By 5 days, the CFSE staining curve has shifted to the left and at least 8 peaks of cell division are present, demonstrating a doubling time, which is equivalent to that found in the cocultured lymphocytes, of approximately 15 hours.