NKT cell isolation, transduction and in vitro expansion

Buffy coats from healthy volunteer blood donors were purchased from the vendor Gulf Coast Regional Blood Center (Houston, Texas, USA). The protocol for the use of purchased buffy coat has been submitted to the institutional IRB and it is considered IRB exempt because it uses deidentified commercial products. Peripheral blood mononuclear cells (PBMCs) were isolated by Lymphoprep (Accurate Chemical and Scientific Corporation) density gradient centrifugation. NKTs were purified from PBMCs as previously described²⁰. Briefly, NKTs they were activated with autologous irradiated PBMCs (40 Gy, RS-2000 Biological System) loaded with α-Galactosylceramide (α-GalCer, 100 ng/ml, Diagnocine LLC) at a PBMC:NKT ratio 10:1 in presence of IL-2 (200 IU/ml, R&D). NKTs were transduced in retronectin coated plates (Takara, 7 µg/ml) on day 5 and further expanded for 10 days in the presence of IL-2 and then used for functional assays.