96 well clear round bottom microplates

The stimulation assay was done as previously described (62 (link)). The assay was set up in 96-well clear round bottom microplates (Corning) with a volume of 200 μl during all incubation steps. T2 cells expressing HLA-A*02:01 were plated at a density of 50,000 cells per well in Iscove’s modified Dulbecco’s medium (Thermo Fisher Scientific) supplemented with 10% FBS (R&D Systems) and penicillin-streptomycin (100 U/ml) and pulsed with 100 mM of the test peptide for 3 hours at 37°C. The cells were then washed and cocultured with Jurkat cells expressing an exogenous TCR of interest (100,000 cells per well) in RPMI 1640 medium (Thermo Fisher Scientific) supplemented with 10% FBS (R&D Systems) and penicillin-streptomycin (100 U/ml) for 16 hours. The next day, the cells were washed with FACS buffer and stained with anti-CD3 (APC, clone OKT3) and anti-CD69 (PE, clone FN50) antibodies for 20 min at 4°C. Cells were washed, resuspended in FACS buffer, and analyzed on the Attune NxT Flow Cytometer (Thermo Fisher Scientific). The data were analyzed using FlowJo (v10) software.