An Optimized Method for Isolating and Expanding Invariant Natural Killer T Cells from Mouse Spleen.

摘要: The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells are therefore characterized as an innate population capable activating and steering adaptive immune responses. development improved techniques for culture expansion murine facilitates study biology in vitro vivo model systems. Here we describe optimized procedure isolation splenic cells. Spleens from C57Bl/6 mice removed, dissected strained resulting cellular suspension layered over density gradient media. Following centrifugation, mononuclear (MNCs) collected CD5-positive (CD5(+)) lymphocytes enriched using magnetic beads. within CD5(+) fraction subsequently stained with αGalCer-loaded CD1d tetramer purified by fluorescence activated sorting (FACS). FACS sorted then initially cultured combination recombinant plate-bound receptor (TCR) stimuli before being expanded presence IL-7. Using this technique, approximately 10(8) can be generated 18-20 days culture, after which they used assays vitro, or transfer experiments mice.