Labeling Murine T Helper Lymphocytes using Radiolabeled Monoclonal Antibodies

Protocolo

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.

Materiais

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