We performed flow cytometry analysis to assess the number of NKT cells in multiple organs. Figure 5a shows representative images in which liver mononuclear cells (MNCs) were stained with anti-TCR αβ and anti-NK1.1 Abs. The proportion of NKT cells in the liver in the α-GalCer group was significantly reduced compared to that in the vehicle group (Fig. 5b). Figure 5c,e and g show representative images in which liver, kidney, and spleen MNCs were stained with anti-TCR αβ Ab and the α-GalCer-loaded CD1d tetramer. The proportions of iNKT cells in the liver, kidney, and spleen in the α-GalCer group were significantly lower than those in the vehicle group (Fig. 5d,f and h).Figure 5Repeated alpha-galactosylceramide (α-GalCer) administration decreases the proportion of NKT cells in multiple organs. (a) Flow cytometry analysis of liver mononuclear cells (MNCs) in the vehicle (left) and α-GalCer (right) groups, stained with anti-TCR αβ and anti-NK1.1 antibodies. In both panels, black squares show NKT cells. Flow cytometry analysis of (c) liver, (e) kidney, and (g) spleen MNCs in the (left) vehicle and (right) α-GalCer groups. Isolated MNCs were double stained with anti-TCR αβ antibody and α-GalCer-loaded CD1d tetramer; invariant NKT (iNKT) cells are shown in black squares. The percentages of (b) whole NKT cells in the liver and iNKT cells in the (d) liver, (f) kidney, and (h) spleen are shown (n = 4 in each group). *p < 0.05 compared with the vehicle group.