Labeling Murine T Helper Lymphocytes using Radiolabeled Monoclonal Antibodies

Published: August 31, 2023

Abstract

Source: Hoffmann, S. H. L., et al., Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo Cell Trafficking by PET/CT. J. Vis. Exp. (2017)

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.

  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.

Declarações

The authors have nothing to disclose.

Materials

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

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Labeling Murine T Helper Lymphocytes using Radiolabeled Monoclonal Antibodies. J. Vis. Exp. (Pending Publication), e21574, doi: (2023).

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