![The presence of α-GalCer in mammalian tissues and fluids. (A) Synthetic β-GalCer (d18:1/16:0 and d18:1/24:1), LPA (O-16:0 and O-18:0), and LPE (P-18:0) were analyzed by HPTLC using C:M:W (65:25:4; vol/vol/vol) followed by staining with copper acetate reagent. Open and closed arrowheads denote the origin and solvent front, respectively. (B) Two clones of human iNKT TCR transfectants (#1 and #2) were stimulated with synthesized α-GalCer (d18:0/16:0) for 16 h and analyzed for CD69 expression. (C) CD1d-transduced B16F10 cells were cultured in RPMI 1640 supplemented with 10% FCS or in RPMI 1640 with 0.6% FCS and 9.4% animal component-free cell culture supplement for 7 days. DN32.D3 cells were co-cultured with those B16F10 cells in the presence of indicated concentrations of α-GalCer (d18:0/16:0) for 16 h and analyzed as in B. (D) HRMS and HRMS/MS chromatograms of HexCer (d18:0/16:0) in the spleen of germ-free mice. The red shadow indicates the peak coincident with synthesized α-GalCer (d18:0/16:0). (E) HRMS and HRMS/MS chromatograms of HexCer detected in human serum by SFC/HRMS/MS. The red shadows indicate the peaks coincident with the theoretical RTs of α-GalCer. The precursor ions as [M+H]+ are m/z 786.6817 and 800.6974, respectively. The HRMS chromatograms were plotted from the theoretical m/z ± 7 ppm of candidate HexCer molecular species. Data are presented as mean ± SD (B and C) and are representative of three independent experiments (A–C). CE, collision energy. Source data are available for this figure: SourceData FS3.](https://cdn.rupress.org/rup/content_public/journal/jem/222/2/10.1084_jem.20240728/1/jem_20240728_figs3.png?Expires=1767647729&Signature=wQA6XVCkZhAZrW-w5ZDJFYF0MpN3Sbo9ZNpnsukwAln41th~6awYQ5uicNY1xmpSVZoo-ESYjkkkprltgmZlcBRjw85XmD8tgeiTkdZDvPyi3ntdE5g8-RzSmsnBHCvXJQUv3XSBP3jnMXhEGoH~3u8ltBXUrZ3nq7gaGS-V-WL-kx2nlgZ6rv1tQK7itkNtrmNFSQDx2M9FgRzWdpqZDtIVIjNWEWqA-BI9U8VCpxW9lwR5X2ZsxLiQyaJ82gxQ8Zj5aE-fSqpOilgUihLkMS7YKyWWfIs7CfWkIUrnMK~3ZwOVC2oE7dgwNUlzAH-vYsrmkAfrun9j3gKhm850NA__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
The presence of α-GalCer in mammalian tissues and fluids. (A) Synthetic β-GalCer (d18:1/16:0 and d18:1/24:1), LPA (O-16:0 and O-18:0), and LPE (P-18:0) were analyzed by HPTLC using C:M:W (65:25:4; vol/vol/vol) followed by staining with copper acetate reagent. Open and closed arrowheads denote the origin and solvent front, respectively. (B) Two clones of human iNKT TCR transfectants (#1 and #2) were stimulated with synthesized α-GalCer (d18:0/16:0) for 16 h and analyzed for CD69 expression. (C) CD1d-transduced B16F10 cells were cultured in RPMI 1640 supplemented with 10% FCS or in RPMI 1640 with 0.6% FCS and 9.4% animal component-free cell culture supplement for 7 days. DN32.D3 cells were co-cultured with those B16F10 cells in the presence of indicated concentrations of α-GalCer (d18:0/16:0) for 16 h and analyzed as in B. (D) HRMS and HRMS/MS chromatograms of HexCer (d18:0/16:0) in the spleen of germ-free mice. The red shadow indicates the peak coincident with synthesized α-GalCer (d18:0/16:0). (E) HRMS and HRMS/MS chromatograms of HexCer detected in human serum by SFC/HRMS/MS. The red shadows indicate the peaks coincident with the theoretical RTs of α-GalCer. The precursor ions as [M+H]+ are m/z 786.6817 and 800.6974, respectively. The HRMS chromatograms were plotted from the theoretical m/z ± 7 ppm of candidate HexCer molecular species. Data are presented as mean ± SD (B and C) and are representative of three independent experiments (A–C). CE, collision energy. Source data are available for this figure: SourceData FS3.