(A and B) MAIT TCR activation assays using NFAT-GFP reporter cells expressing mouse MAIT TCR and MR1. Reporter cells were stimulated with vehicle control, 5-OP-RU, and analogs of CA7S (1); cholic acid 3-sulfate (CA3S: 2), cholic acid 12-sulfate (CA12S: 3), cholic acid (CA: 4), taurocholic acid (TCA: 5), deoxycholic acid (DCA: 6), taurocholic acid 3-sulfate (TCA3S: 7), taurocholic acid 7-sulfate (TCA7S: 8), deoxycholic acid 3-sulfate (DCA3S: 9), lithocholic acid 3-sulfate (LCA3S: 10), and taurolithocholic acid 3-sulfate (TLCA3S: 11). Because of their inherent cell toxicity as surfactants, several bile acids were added at lower concentrations (4, 5, 6, 10, and 11). Some water-insoluble bile acids (4, 5, 6, and 10) were coated on plates as described (81). Compound 11 was added as dimethyl sulfoxide solution because of its insolubility in both water and organic solvents. NFAT-GFP (A) and MR1 (B) expressions were evaluated at 20 and 6 hours after stimulation, respectively. (C) Structural formula of bile acid analogs related to (A) and (B). (A and B) Data are presented as individual values of duplicate assays and are representative of more than two independent experiments.