Flow cytometer–based proliferation assay with BrdU to detect proliferation of cells from different origins simultaneously.

More than 100 days after transplantation, 1.25 × 10⁶spleen cells per well were cultured with immobilized anti-CD3 in a flat-bottom 48-well culture plate at 37°C and 5% CO₂ for 48 hours. A final concentration of 30 μM BrdU was added 24 hours before harvest. After culture, the cells were transferred into 6-mL tubes and washed once. The cells were first stained with anti–surface marker antibodies, washed once, and then resuspended in 0.5 mL FACS Permeabilizing Solution for fixation and permeabilization. After 3 hours' incubation at 4°C, cells were washed twice and stained with anti-BrdU antibody (1 μg/mL) in the presence of DNAse I at a final concentration of 4 mg/mL for 30 minutes at room temperature. The stained cells were analyzed by means of a Coulter EPICS XL flow cytometer equipped with System II software.