Nur77-GFP Jurkat 76 TCRα–β– cell line

To characterize the functionality of TCRαβ or γδ clones, we established the Nur77-GFP Jurkat 76 TCRα^–β^– cell line (NJ76 cells). After linearization of a Nur77-GFP BAC clone (constructed based on pTARBAC)³⁹ by mixing 10 μg BAC DNA, 2 μl 10× reaction buffer, 10 units of PI-SceI restriction enzyme (New England Biolabs), and nuclease-free water to make the volume up to 20 μl with incubation at 37 °C for 3 hours and inactivation at 65 °C for 20 minutes, we added 80 μl of nuclease-free water, 15 μl of sterile sodium acetate (3M, pH 7.0), and 300 μl of ethanol to the reaction mixture, and centrifuged at 12,000g for 30 minutes at 4 °C. The resulting DNA pellet was washed with 75% ethanol, dried in the air, and resuspended by Tris-EDTA buffer (pH 8.0). We used the Neon Transfection System (Invitrogen) following the manufacturer’s instruction to transfect the linearized BAC DNA into the human Jurkat 76 TCRα^–β^– cell line (2 × 10⁷ cells/ml, 100 μl), with three pulses with a voltage of 1,350V and a width of 10 ms. Cells were then cultured in complete-RPMI 1640 medium containing 500 μg/ml Geneticin (Invitrogen) for selection.