MR1-Tetramers Staining

Human MR1-5-OP-RU labeled with BV421 or PE and MR1-tetramers were generated as described previously (2, 51). Briefly, refolded and purified empty carboxy-terminal cysteine-tagged-MR1 was loaded with a 136 molar excess of synthetic 5-OP-RU for 4 h at room temperature in the dark. For co-staining with MR1-tetramers, ~1 × 10⁵ cells were stained with MR1-5-OP-RU tetramer at 20 μg/ml for 40 min at room temperature in the dark. Cells were then washed and stained with anti-CD3, CD161, and TCRα7.2 for 30 min at 4°C. Cells were then washed once with 2 ml of PBS wash (2% fetal bovine serum in PBS) and resuspended in 150 ml of FACS fix (2% glucose and 1% paraformaldehyde in PBS) before acquisition of data on a BD LSR-Fortessa.