Isolation and Identification of MAIT Cells from Human Blood

Peripheral venous blood samples were collected in heparin-containing tubes, and PBMCs were isolated by density-gradient centrifugation using Ficoll-Paque Plus solution (Amersham Bioscience, Uppsala, Sweden). MAIT cells were identified phenotypically as CD3+TCRγδ-Vα7.2+CD161^high by flow cytometry as previously described [10 (link), 24 (link), 45 (link)]. Total lymphocyte numbers were measured by Coulter LH750 automatic hematology analyzer (Beckman Coulter, Miami, FL). Absolute numbers of MAIT cells were calculated by multiplying the MAIT cell percentages by the CD3+γδ- T cell percentages and the total lymphocyte numbers (per microliter) in peripheral blood. For sorting of MAIT cells, PBMCs were stained with PE-conjugated anti-CD3, FITC-conjugated anti-TCR γδ, APC-conjugated anti-TCR Vα7.2 and PE-Cy5-conjugated anti-CD161 and sorted to obtain CD3+TCRγδ-Vα7.2+CD161^high MAIT cells using a FACS Aria I sorter (BD Biosciences, Mountain View, CA). MAIT cells were isolated at purities of > 98%.