Fig. 2.

Cells overexpressing CD1d bind to NGcGM3. a Flow cytometry analysis of NGcGM3 binding to the surface of the C1RCD1d cell line. Cells were incubated for 24 hs with empty liposomes (left panel) or with NGcGM3-loaded liposomes (right panel) followed by staining with a mAb (14F7, referred as aNGcGM3 in the figure, black line) or isotype-matched control mAb (gray-filled histogram). Detection of 14F7 bound to C1RCD1d cell surface was evidenced by PE-conjugated aIgG1 staining. Data are representative of three independent experiments. b Flow cytometry analysis of NGcGM3 binding to the surface of the C1RCD1d and C1R cell lines. Staining as in a with an untransfected C1R cell line. c Effect of PFA treatment on NGcGM3 binding to C1RCD1d cells. Control (untreated, left panel) and PFA-treated cells (right panel) were incubated with NGcGM3-loaded liposomes, which was followed by staining, as in a, for either NGcGM3 binding (black line) or isotype-matched controls (gray-filled histogram). Data are representative of three independent experiments. d Effect of PFA treatment on CD1d expression evaluated by C1RCD1d. The black line indicates cells that were fixed with 4 % PFA for 20 min at 4 °C and washed with PBS with 10 % FCS. Finally, they were stained with PE-conjugated aCD1d (clone CD1d42). The gray-filled histogram indicates untreated cells. Data are representative of three independent experiments