Neonatal or adult NKT cells were purified and activated under conventional Th0 (medium alone; NKT0), Th1 (NKT1), or Th2 (NKT2) polarizing conditions as described in the methods and expanded in IL-2-containing media.
These cells were re-stimulated for 6–8 hours using DCs pulsed with α-GalCer (100 ng/ml), or alternatively, anti-CD3 and anti-CD28, in the presence of GolgiStop (BD) and were then intracellularly stained with antibodies against IL-4 (PE) and IFN-γ (APC).
NKT cells were also co-stained with anti-Vβ11 (FITC) to exclude contaminating non-NKT cells.
For adult NKT cells, CD4 expression was also assessed by cell surface staining (CD4-biotin followed by strepavidin-PerCP-Cy5.5) to differentiate CD4+ from CD4− NKT cells.
One representative donor out of 5 is shown.