For expanding human NKT cells from freshly isolated PBMC:

Mix iGb3 at 100 ng/ml with PBMC and culture for 5 days.

For stimulating mouse NKT cells:

The bone marrow derived dendritic cells is the most efficient cell type that present iGb3. We use 50,000 to 250,000 of BMDCs pulsed overnight with 100 ng/ml of iGb3, and 50,000 freshly purified NKT cells as a responder. We use the tetramer sorting to purify the fresh NKT cells from spleen, and before mixing them with BMDCs we rest them in culture medium for 12 hours and then wash with medium. The reason is that the sorting with tetramer seems to activate the cells and give you some background (sometimes up to 200 pg/ml of IFN-g). Please avoid to use MACS technique to do the sorting, in our hands they kill lots of NKT cells.

Alternatively, the NKT cell hybridomas (DN32D3 by Bendelac lab or N382C11 by Dr. Kyoko Hayakawa) can be used to detect the ligand.