Methods for detection, isolation and culture of mouse and human invariant NKT cells

This protocol describes methods to identify, purify and culture CD1d restricted invariant natural killer T ( i NKT) cells from mouse tissue or human blood samples. The methods for identification and purification of i NKT cells are based on the interaction between i NKT cell receptor and its ligand. The i NKT cell receptor is composed of the invariant Vα14Jα18/Vβ8.2 in mice or Vα24Jα18/Vβ11 in humans and is expressed only on i NKT cells but not on conventional T cells. The i NKT cell antigen receptor in both species recognizes α-galactosylceramide (α-GalCer) presented by the MHC class I-like CD1d. Thus, α-GalCer-loaded CD1d dimer can be used for analysis and purification by fluorescence-activated cell sorting (FACS). Isolation of 1 × 10 6 purified i NKT cells from mouse thymus, spleen or liver requires 5–6 mice and takes 1–2 h for mononuclear cell preparation from mouse tissues, 1.5 h for enrichment by magnetic beads and 4 h for detection and purification of the i NKT cells by FACS. In the case of isolation of human peripheral blood mononuclear cells (PBMCs) from whole blood, it takes 2 h and requires 5 ml of blood to obtain 5 × 10 6 PBMCs, which contain 500–25,000 i NKT cells.