Flow Cytometric Analysis of MAIT Cell Effector Molecules

Cell-surface staining was performed using directly-conjugated antibodies and cells were fixed in Cytofix/Cytoperm or in Transcription Factor Fixation/Permeabilization buffer (both from BD Biosciences) as appropriate. Intracellular staining was performed in Perm/Wash or Transcription Factor Perm/Wash buffer as appropriate (both from BD Biosciences). Samples were acquired on an LSRFortessa flow cytometer (BD Biosciences) equipped with 405, 488, 561, and 639 nm lasers. Single-stained polystyrene beads (BD Biosciences) were used for compensation purposes. Software-based compensation was performed using the compensation platform in FlowJo software version 9.6 (Tree Star, Ashland, OR, USA). Distribution of effector molecules expressed by MAIT cells was compared using SPICE software version 5.35.^{48 (link)} Antibodies used in the experiments are listed in Supplementary Table 2.