Apc anti mouse tcrβ chain clone h57 597

10⁷ Jurkat cells per transfection were centrifuged at 250 × g for 5 min, resuspended in 5 mL of OptiMEM (Gibco), and incubated at 20 °C for 8 min. Cells were centrifuged as before, resuspended in 400 µl of OptiMEM and 20 µg HA1.7 plasmid, and transferred to a 4-mm electroporation cuvette (Bio-Rad). Cells were incubated for 8 min before pulsing exponentially with 250 V, 950 µF, and ∞ ohms resistance on a Bio-Rad GenePulser Xcell with PC and CE modules. After an 8 min recovery period, cells were rescued with 10 mL of prewarmed Jurkat culture media (RPMI 1640 + 10% FBS + 100 U/mL penicillin-streptomycin) and kept at 37 °C, 5% CO₂.
In vitro stimulation of HA1.7 TCR-transfected Jurkat cells was performed 12–16 h after transfection. αCD3/αCD28 microbeads or titrations of nanoscale DR1 HA peptide exchanged and DR1 CLIP unexchanged aAPCs were incubated at 37 °C with 5 × 10⁴ transfected Jurkat T cells per stimulation in Jurkat culture media. At 24 h post-transfection, samples were washed and stained for 15 min at 4 °C in FACS Wash Buffer with APC anti-mouse TCR β chain clone H57-597 (BioLegend) and FITC anti-human CD69 clone FN50 (BioLegend) to detect the HA1.7 TCR and activation, respectively. Cells were then washed again and analyzed on a BD FACSCalibur Flow Cytometer.