Anti cd8 pecy7

Anti-CD127-PECy7, anti-CD25-PECy5, anti-CD45RA-APCH7, anti-CD19-FITC, and anti-CD3-APCCy7 were purchased from BD Biosciences (San Jose, CA, USA). Anti-CD4-PE and anti-CD8-PECy7 were purchased from Tonbo Biosciences (San Diego, CA, USA). Anti-FOXP3-AlexaFluor647 was purchased from Beckman Coulter (Brea, CA, USA). Carboxy fluorescein succinimidyl ester (CFSE) was purchased from Life Technologies (Eugene, OR, USA).
For intracellular staining, FOXP3/Transcription Factor Staining Buffer Set (eBiosciences, San Diego, CA, USA) was used. All staining were performed in RPMI 1640 (Gibco, Carlsbad, CA, USA) with 10% fetal bovine serum (FBS), 2 mM glutamine, 10 mM HEPES, and 10 mM antibiotic-anti-mycotic (Gibco).

For phenotypic analysis, single cell suspensions were stained as previously described 36. For ex vivo Treg cell analysis, anti‐CD25‐PECy5, anti‐Helios‐FITC, anti‐CD8‐PE (from Biolegend, San Diego, CA, USA), anti‐CD4‐APC‐AF750 (from Thermo Fisher Scientific), and anti‐FoxP3‐APC (from eBiosciences, San Diego, CA, USA) were used. For in vitro induced Treg analysis, Zombie Aqua fixable dye, anti‐CD4‐APC and anti‐CD25‐PECy5 from Biolegend were used. For ex vivo DC analysis, cells were blocked with purified anti‐CD16/32, followed by staining with Zombie Aqua, anti‐I‐A/I‐E‐AF488, anti‐CD11c‐AF700, anti‐CD80‐PECy5 (from Biolegend), anti‐CD3‐PE, anti‐TER119‐PE, anti‐CD11b‐VF450, anti‐CD86‐APC, anti‐CD8‐PECy7 (from Tonbo Biosciences, San Diego, CA, USA), anti‐CD19‐PE, anti‐CD49b‐PE, streptavidin‐APCCy7 (from BD Biosciences, San Jose, CA, USA), anti‐CD103‐biotin and anti‐PD‐L1‐PerCP‐eFluor710 (from eBiosciences) were used.
For in vivo transfer experiments, anti‐CD45.1‐AF700, anti‐CD4‐FITC, anti‐CD25‐PECy5, streptavidin‐BV605 (from Biolegend), anti‐Vβ5‐biotin (from BD Biosciences), and anti‐FoxP3‐APC (from eBiosciences) were used for staining ovalbumin (OVA)‐specific T cells.
Samples were acquired in an Attune Acoustic Focusing Flow Cytometer (Thermo Fisher Scientific) and analyzed using flowjo 10.0 software (Tree Star Inc., Ashland, OR, USA).