![Detection of α-GalCer in serum and bile. (A) DN32.D3 cells were stimulated with synthesized α-GalCer (d18:0/16:0) or α-GalCer (t18:0/26:0) (KRN7000) for 16 h and analyzed for CD69 expression. (B) Lipid extracts from bovine bile were separated into five fractions by open column chromatography and analyzed by HPTLC using C:M:W (65:25:4; vol/vol/vol) followed by staining with copper acetate reagent. (C) CD1d−/− or CD1d-transduced DN32.D3 cells were stimulated for 16 h with each fraction of bovine bile lipids separated in B and analyzed as in A. α-GalCer (t18:0/26:0) was used as a positive control. (D) The C:M = 19:1 fraction of bovine bile lipid in B was separated into 16 subfractions by HPTLC (left) and used for stimulation as in C (right). α-GalCer (t18:0/26:0) was used as a positive control. (E) HRMS and HRMS/MS chromatograms of HexCer (d18:0/16:0) in bovine bile. (F and G) The HRMS spectra (F) and HRMS/MS spectra (G) of candidates for α-GalCer (d18:0/16:0) in bovine bile detected in D. From top to bottom, collision energy settings are −10 eV, −20 eV, −30 eV, and −40 eV (G). (H) HRMS chromatograms of HexCer (d18:0/16:0) and the HRMS/MS spectra of candidates for α-GalCer (d18:0/16:0) in bovine bile spiked with synthesized α-GalCer (d18:0/16:0). The area under the curve value ratio of the peak coincident with synthesized α-GalCer (d18:0/16:0) is 4.04 × 106:7.22 × 106. From left to right, collision energy settings are −10 eV (+), −20 eV (++), −30 eV (+++), and −40 eV (++++). The structures of product ions are shown. Open and closed arrowheads denote the origin and solvent front, respectively (B and D). The mass error tolerance of the precursor ion (m/z 702.5878 as [M+H]+) was <7 ppm (G and H). The HRMS/MS of precursor-product ion pair: m/z 702.5878 to 540.5350, 522.5245, 284.2948, and 266.2842, respectively (G and H). The red shadow indicates the peak coincident with synthesized α-GalCer (d18:0/16:0) (E and H). Data are presented as mean ± SD (A, C, and D) and are representative of three independent experiments (A–H). CE, collision energy. Source data are available for this figure: SourceData F3.](https://cdn.rupress.org/rup/content_public/journal/jem/222/2/10.1084_jem.20240728/1/jem_20240728_fig3.png?Expires=1767647719&Signature=3sG8c2ugdwcs13mmx7n5JqfRvRsMtnLkqP4ZW8sLS9OO5xcqmTmAYt0HbMEaY4GrTuYV0i32Nhui7SKialS~5FAGB--HDz83fb9Y0tJDooWJVo46q2abC8B5JdUvbm~hecdT51cftD2NyM2HHpAfgQui-MbRKCZQzB24IBMnqNPuVOPCwQfs4VZChShrVESg~Xd84BPRH4XbUtNdYWTmX0hxjBcNcTWSiuayGwA85CxnPFKAGdMUk9kjlJqb6ET-ZX74wxjqXa3F61TlwQjpvuMkUssvfuNU~9f6nW1PUwt6C2j0y6d5O8VQYTvSY-tkgrEb1qIFeYfApYMVkB3AOQ__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Detection of α-GalCer in serum and bile. (A) DN32.D3 cells were stimulated with synthesized α-GalCer (d18:0/16:0) or α-GalCer (t18:0/26:0) (KRN7000) for 16 h and analyzed for CD69 expression. (B) Lipid extracts from bovine bile were separated into five fractions by open column chromatography and analyzed by HPTLC using C:M:W (65:25:4; vol/vol/vol) followed by staining with copper acetate reagent. (C) CD1d−/− or CD1d-transduced DN32.D3 cells were stimulated for 16 h with each fraction of bovine bile lipids separated in B and analyzed as in A. α-GalCer (t18:0/26:0) was used as a positive control. (D) The C:M = 19:1 fraction of bovine bile lipid in B was separated into 16 subfractions by HPTLC (left) and used for stimulation as in C (right). α-GalCer (t18:0/26:0) was used as a positive control. (E) HRMS and HRMS/MS chromatograms of HexCer (d18:0/16:0) in bovine bile. (F and G) The HRMS spectra (F) and HRMS/MS spectra (G) of candidates for α-GalCer (d18:0/16:0) in bovine bile detected in D. From top to bottom, collision energy settings are −10 eV, −20 eV, −30 eV, and −40 eV (G). (H) HRMS chromatograms of HexCer (d18:0/16:0) and the HRMS/MS spectra of candidates for α-GalCer (d18:0/16:0) in bovine bile spiked with synthesized α-GalCer (d18:0/16:0). The area under the curve value ratio of the peak coincident with synthesized α-GalCer (d18:0/16:0) is 4.04 × 106:7.22 × 106. From left to right, collision energy settings are −10 eV (+), −20 eV (++), −30 eV (+++), and −40 eV (++++). The structures of product ions are shown. Open and closed arrowheads denote the origin and solvent front, respectively (B and D). The mass error tolerance of the precursor ion (m/z 702.5878 as [M+H]+) was <7 ppm (G and H). The HRMS/MS of precursor-product ion pair: m/z 702.5878 to 540.5350, 522.5245, 284.2948, and 266.2842, respectively (G and H). The red shadow indicates the peak coincident with synthesized α-GalCer (d18:0/16:0) (E and H). Data are presented as mean ± SD (A, C, and D) and are representative of three independent experiments (A–H). CE, collision energy. Source data are available for this figure: SourceData F3.