Anti cd25 bv650

We cultured 2,000–5,000 MAIT cells from MAIT cells expanded in RPMI 1640 with 10% FBS or 2% Phx with 10,000–25,000 THP-1 cells to serve as APCs in RPMI with 10% FBS without cytokines overnight. We then stimulated the coculture of MAIT cells and THP-1 with 10 MOIs of strain 1100–2 fixed E. coli for 20 h and blocked extracellular transport with brefeldin A for a total of 4 h. For extracellular staining, we used Zombie ultraviolet fixable viability dye (BioLegend), anti-CD3-BUV395 (BD Biosciences), anti-CD8-BV605 (BioLegend), anti-CD4-BUV496 (BD Biosciences), anti-Vα7.2-PE-Cy7 (BioLegend), anti-LAG-3-BV786 (BioLegend), anti-CD69-BUV563 (BD Biosciences), anti-CD161-PE-Dazzle-594 (BioLegend), anti-TIM-3-BV421 (BioLegend), anti-CD25-BV650 (BD Biosciences), and anti-human PE-MR1–5-OP-RU tetramer (NIH Tetramer Core Facility). The cells were fixed and permeabilized using Foxp3/transcription factor kit (eBioscience) and stained intracellularly with anti-granzyme-B Alexa Fluor 700 (BioLegend), anti-TNF-α eFluor 450 (Invitrogen), and anti-IFN-γ-FITC (BioLegend). Sample data were acquired with a five-laser Cytek Aurora flow cytometer (Cytek) and analyzed using FlowJo software version 10 (BD Biosciences).