Anti cd8α pacific blue

All mAbs used for the study are listed in Table S3. Single cell suspensions (2–3 × 10⁶) were incubated with Ghost Voilet 510 viability dye (Tonbo Bioscience) in PBS containing 50 nM Dasatinib (LC laboratories) to differentiate between live/dead cells. After washing cells with FACS buffer (2% v/v FBS and 50 nM Dasatinib in PBS) once, cells were incubated in FACS buffer containing 0.2 μg anti-CD16/CD32 mAb for 15 min on ice to block mAbs from binding to Fc receptors. Cells were then incubated in the dark with fluorochrome-conjugated mAbs to detect surface markers. After 45 min, cells were washed twice with FACS buffer. Surface-stained cells were incubated with 0.25–0.5 μg/ml pB7.2 tetramers in PBS containing 50 nM Dasatinib. For the screening study, we adopted a dual colour encoding strategy as described^{23 (link)}. With five fluorochromes, we detected 10 different specificities in a single reaction. Flow cytometric data were acquired using FACSCanto II (BD Biosciences) and FACS (.fcs) files were analysed with FlowJo software (Tree Star).
Phenotypic analysis of CD8⁺ T cells was performed by co-staining with p/B7.2 tetramers and anti-CD8α-Pacific Blue, -B220-FITC, -CD44-APC (clone IM7), -CD62L-APC-Cy7 (clone MEL-14), -CD127-PE (clone SB/14; all from BD Bioscience) or anti-KLRG-1-APC (clone 2F1; eBioscience).