Overview
In this video, we demonstrate the intracellular radiolabeling of T helper cells using radioactive isotope-conjugated monoclonal antibodies for non-invasive positron emission tomography imaging.
Protocol
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.
- 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.
- 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.
- 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.
- 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.
- 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.
- Count viable cOVA-TH1 cells in an appropriate dilution with trypan blue staining.
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Materials
| Name | Company | Catalog Number | Comments |
| 64Cu work-up module | WKL | Custom made | 64Cu production |
| Dose calibrator | Capintec | CRC-25R | |
| PETtrace cyclotron | General Electric Medical Systems | ||
| DOTA-NHS | Macrocyclics | B-280 | |
| Anti-cOVA-TCR antibody (KJ1-26) | Isolated from hybridoma cell culture | DOTA-conjugation | |
| Na2HPO4 | Sigma-Aldrich | 71633 | DOTA-conjugation |
| H+ Chelex 100 | Sigma-Aldrich | C7901 | DOTA-conjugation |
| Amicon Ultra-15 filter unit | Merck Millipore | UFC910008 | DOTA-conjugation |
| Rotipuran ultrapure water | Carl Roth | HN68.3 | DOTA-conjugation |
| Ammonium acetate | Sigma-Aldrich | 32301 | DOTA-conjugation |
| PBS | University Tuebingen | DOTA-conjugation | |
| Micro Bio-spin P-6 column | Bio-Rad Laboratories | 7326221 | DOTA-conjugation |
| Sodium citrate | Sigma-Aldrich | 71497 | DOTA-conjugation |
| Cyclone Plus PhosphorImager | Perkin-Elmer | L2250116 | DOTA-conjugation |
| DMEM | Merck Millipore | 102568 | Ingredient for T cell medium |
| FCS | Merck Millipore | S0115/1004B | Ingredient for T cell medium |
| Sodium pyruvate | Merck Millipore | L0473 | Ingredient for T cell medium |
| MEM-amino acids | Merck Millipore | K0293 | Ingredient for T cell medium |
| HEPES | Merck Millipore | L 1613 | Ingredient for T cell medium |
| Penicillin/Streptomycin | Merck Millipore | A2212 | Ingredient for T cell medium |
| 0.05 mM 2-β-mercaptoethanol | Sigma-Aldrich | M3148 | Ingredient for T cell medium |
| DO11.10 mice | In-house breeding | TH1 cell culture | |
| DPBS | Gibco | 14190144 | TH1 cell culture |
| Cell strainer 40 µm | Corning | 352340 | TH1 cell culture |
| ACK Lysing Buffer | Lonza | 10-548E | TH1 cell culture |
| CD4 MicroBeads, mouse | Miltenyi Biotech | 130-097-145 | TH1 cell culture |
| QuadroMACS separator | Miltenyi Biotech | 130-090-976 | TH1 cell culture |
| LS column | Miltenyi Biotech | 130-042-401 | TH1 cell culture |
| Anti-CD4 antibody (Gk1.5) | Isolated from hybridoma cell culture | TH1 cell culture | |
| Anti-CD8 antibody (5367.2) | Isolated from hybridoma cell culture | TH1 cell culture | |
| Anti-rat antibody (MAR18.5) | Isolated from hybridoma cell culture | TH1 cell culture | |
| Rabbit complement MA | tebu-Bio | CL3221 | TH1 cell culture |
| Anti-IL-4 antibody (11B11) | Isolated from hybridoma cell culture | TH1 cell culture | |
| cOVA 323-339-peptide | EMC-micro-collections | Custom order | TH1 cell culture |
| CPG1668-oligonucleotides | Eurofins MWG Operon | Custom order | TH1 cell culture |
| IL-2 | Novartis | 65483-116-07 | TH1 cell culture |
| 96-well plates | Greiner | 655180 | TH1 cell culture |
| 24-well plates | Greiner | 662160 | TH1 cell culture |
| Cell culture flask | Greiner | 660175 | TH1 cell culture |
| 48-well plates | Greiner | 677 180 | Cell labeling |
| Gammacell 1000 | Best Theratronics | Via inquiry | |
| Gulmay RT225 | Gulmay | Via inquiry | |
| Trypan blue | Merck Millipore | L6323 | In vitro evaluation |
