One million CD19-tPSMA(N9del) CAR T cells were incubated with 74 MBq of [18F]DCFPyL in 1 ml of PBS at 37°C for 30 min, followed by two washes with PBS. Stained CD19-tPSMA(N9del) CAR T cells were plated in 384-well plates with serial dilutions from 40,000 to 200 cells in 20 μl of PBS. The plate was scanned with the SuperArgus small-animal PET/CT scanner (Sedecal, Madrid, Spain) at an energy window of 250 to 700 keV for 5 min (n = 8). Images were processed with Amide software (Dice Holdings Inc., NY, USA). A standard curve was generated with total voxel values per number of cells. [18F]DCFPyL was obtained from the Johns Hopkins University PET Center as the residual tracer from the clinical preparation, the synthesis of which has been previously described (29).

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