Anti tcr α β pe cy7

For experiments using F5-Jurkat, 1G4-Jurkat, MART1-K562 and NYESO-K562 cells, co-incubations were set up in 96-well U-bottom plates at a ratio of 5:1 Jurkat to K562 (300,000 cells per well) and co-incubated for 45 min at 37 °C. Ratios for co-incubation experiments involving SCT-Jurkat cells and TCR-K562 cells are indicated in the text and figure legends. The co-incubation was then centrifuged for 5 min at 1,500 r.p.m. and the medium was aspirated by vacuum. The cell pellets were resuspended with cold PBS solution containing 2 mM EDTA and then centrifuged. Cells were stained with different antibodies at 4 °C for 20 min, washed twice, and then analyzed by flow cytometry using MACSQuant Analyzer 10 (Miltenyi Biotec). The following antibodies were used to detect expression of surface markers: anti-CD3-PE, anti-CD8-BV510, anti-TCRαβ-PacificBlue, anti-TCRαβ-PE/Cy7, anti-LNGFR-PE, anti-LNGFR-APC, anti-HLA-A2-PacificBlue, anti-HLA-A2-BV510, and anti-muTCRa β-PE/Cy7 (all from Biolegend). Flow cytometry gating strategies are described in the Supplementary Note.

The capture of activated CD4⁺ T cells was performed as previously reported^{19 (link)}. Briefly, PBMCs were thawed in complete RPMI 1640 medium at 2 × 10⁶ cells/ml and recovered 12 hours before stimulation. PBMCs were stimulated with an Mtb lysate (10 μg/ml) for 12 hours in the presence of 1 μg/ml purified anti-CD49d antibody and anti-CD154-BV421. After stimulation, cells were harvested and stained with antibodies to various cell surface markers, including anti-CD3-Alexa700, anti-TCR α/β-PE/Cy7, anti-CD4-PerCP/Cy5.5, anti-CD8-BV605, anti-CD69-APC/Cy7 abs from BioLegend; purified anti-CD49d and anti-CD154-BV421 abs from BD Biosciences. Dead cells were stained using LIVE/DEAD™ Fixable Aqua Dead Cell Stain Kit from Thermo Fisher scientific. Activated CD4⁺ T cells were either single-cell sorted into 96-well plate for single cell TCRα/β sequencing or bulk sorted into an individual tube for TCRβ library preparation.