Encyclopedia of Experiments: Immunology
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Transcript
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.
