Labeling Murine T Helper Lymphocytes using Radiolabeled Monoclonal Antibodies

1. cOVA-TH1 Cell Radiolabeling

NOTE: The ⁶⁴Cu-DOTA-KJ1-26-mAb will be applied to cultured cOVA-TH1 cells to enable intracellular radioactive labeling.

1. For cOVA-TH1 cell radiolabeling, draw 37 MBq of the radiolabeled ⁶⁴Cu-DOTA-KJ1-26-mAb in a syringe without dead volume using a dose calibrator. Add 1 mL saline to receive a 37 MBq/mL solution.
2. Suspend cOVA-TH1 cells in the medium at 2 x 10⁶ cells/mL and add 0.5 mL of the cell suspension to each well in a 48-well plate.
3. Add 20 µL of the freshly prepared 37 MBq/mL ⁶⁴Cu-DOTA-KJ1-26-mAb solution to each well of the 48-well plate. The final ratio for the labeling is 0.7 MBq per 10⁶ cells in a volume of 520 µL. Incubate at 37 °C and 7.5% CO₂ for 30 min.
4. After incubation, carefully resuspend the cells in each well and transfer the cell suspension from the 48-well plate into a 50 mL screw cap tube. To minimize cell loss, rinse each well with a pre-warmed medium.
5. Centrifuge the cOVA-TH1 cell suspension at 400 x g for 5 min, discard the supernatant, and resuspend the cells in 10 mL prewarmed PBS. Remove an aliquot of the cell suspension for cell counting. Repeat the washing step.
6. Count viable cOVA-TH1 cells in an appropriate dilution with trypan blue staining.