In Vitro and in Vivo Cytokine Production.

For in vitro cytokine production, total thymocytes were placed in 24-well plates, at a concentration of 10 × 10⁶ cells per milliliter in RPMI medium, and stimulated for 72 h with 125 ng/mL α-galactosylceramide (KRN7000). Phorbol 12-myristate 13-acetate (PMA) (50 ng/mL), ionomycin (500 ng/mL), and Golgiplug (1 ng/mL) were added to the cells for the final 5 h. For in vivo stimulation of iNKT cells, mice were injected intraperitoneally with 4 μg α-galactosylceramide. Four hours later, splenocytes were collected, stained intracellularly for cytokines, and analyzed by flow cytometry.