Figure 62.

Gating on human blood NKT cells. (A) Lymphocytes are distinguished amongst PBMCs based on their relative FSC-A and SSC-A intensities. Single cells are then isolated by their relationship between FSC-H versus FSC-A, and SSC-W versus SSC-A. To remove any non-specific or TCR-independent CD1d-tetramer staining, dead cells are removed from analysis based on their uptake of LIVE/DEAD^™ Fixable Near-IR viability dye. Monocytes and B cells are also excluded based on their CD14 and CD19 expression respectively. (B) The frequency of circulating Type I NKT cells, as determined by co-staining for CD3ε and α-GalCer (PBS-44)-loaded CD1d-tetramer (left) in relation to a vehicle control CD1d-tetramer (right). (C) The frequency of iNKT cells was assessed by co-staining with 6B11 and anti-Vβ11. (D) Co-staining with anti-Vα24 and anti-Vβ11, which non-exclusively enriches for iNKT cells.