This article describes a method for radiolabeling murine T helper lymphocytes that express a T cell receptor specific for chicken ovalbumin peptide. The process involves using radiolabeled monoclonal antibodies to facilitate the internalization of the TCR complex into the cytoplasm of the lymphocytes.
To radiolabel murine T helper, Th lymphocytes carrying a T cell receptor, TCR, specific for chicken ovalbumin peptide, take a tube containing radiolabeled monoclonal antibodies, mAbs, with the desired radioactive activity.
The mAb comprises a murine mAb specific for the TCR expressed on the murine Th lymphocyte membrane. A chelator conjugates the mAb and a positron-emitting copper isotope, forming a stable complex.
Dilute the radiolabeled mAbs with saline. Next, add the diluted radiolabeled mAbs to a multi-well plate containing a Th lymphocyte suspension. Briefly incubate.
Within the culture, the radiolabeled mAbs bind to the TCRs on the Th lymphocyte membranes. Post-incubation, transfer the cell suspension to a fresh tube.
Centrifuge. Discard the supernatant containing unbound radiolabeled antibodies. Resuspend the Th lymphocytes in medium and transfer into a multi-well plate. Incubate for an extended duration.
The radiolabeled mAb-TCR binding causes adaptor protein recruitment to the cytoplasmic side of the plasma membrane, to which clathrin triskelions bind and form a clathrin-coated pit.
Accessory proteins help pinch off the vesicle from the plasma membrane, facilitating internalization of the radiolabeled mAb-TCR complex into the cytoplasm. Eventually, TCRs are re-expressed on the Th lymphocyte membranes.
The radiolabeled Th lymphocytes are ready for further experiments.
For chicken ovalbumin-specific TH1 cell radiolabeling, first use a dose calibrator to draw 37 megabecquerels of the T cell-specific radioactive antibody of interest, into a syringe without dead volume.
Next, dispense the antibody into a reaction cup, and measure and subtract the remaining amount of reactivity in the syringe from the amount that was drawn. Then, add one milliliter of saline to the cup to generate a 37-megabecquerel-per-milliliter solution.
Next, add two times 10 to the sixth OVA-TH1 cells in 0.5ml of complete medium to each well of a 48-well plate, followed by 20 microliters of the radiolabeled antibody solution. After 30 minutes in a radiation-safe 37-degrees Celsius and 7.5% carbon dioxide cell culture incubator, pool the radiolabeled cells in a 50-milliliter conical tube for two washes in 10 milliliters of 37 degrees Celsius PBS.
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