Characterization of glycolipid Glc-DAG purified from S. pneumoniae.

(A) NFAT-GFP reporter cells (4 x 10⁴ cells/well) were incubated with live S. pneumoniae at MOI 0.2, 2, 20 and 200 for 18 h followed by determination of the percentage of GFP-expressing reporter cells by flow cytometry. (B) FcRγ or Mincle+FcRγ expressing NFAT-GFP reporter cells (4 x 10⁴ cells/well) were incubated with aqueous or C:M fraction of S. pneumoniae lysates for 18 h, followed by determination of GFP-expressing reporter cells by flow cytometry. (C) HPTLC result of HPLC fractionated C:M portion of pneumococcal lysates with putative ligand indicated by an arrow head (copper acetate stain). (D) FcRγ (white bars) or Mincle + FcRγ (black bars) NFAT-GFP reporter cell assay of HPLC fractions obtained from scratched and purified HPTLC bands of S. pneumoniae lysates shown in (C). Experiments were repeated three times with similar results. See also S2 Fig. (E-H) Effect of TDM (E), or S. pneumoniae-derived Glc-DAG (F), or synthetic Glc-DAG (C14:0/C14:0, (G), or C18:0/C18:0, (H)) to trigger GFP reporter activation in Mincle/FcRγ (black dots), or FcRγ only (white dots) expressing NFAT-GFP reporter cells.