T cell purification and activation

This protocol details the purification and activation of Mouse Naïve CD8+ T Cell from mouse spleens using a purification kit. The cells are then activated by culturing them in plates coated with anti-CD3 antibodies and adding soluble anti-CD28 antibodies. The activation process occurs over 72 hours, with IL-2 added after the first 24 hours to support T cell proliferation and survival. The isolation kit from STEMCELL is designed to isolate naïve CD62L+CD44-CD8+ T cells from single-cell suspensions of splenocytes by negative selection. Unwanted cells are targeted for removal with biotinylated antibodies that are directed against non-naïve CD8+ T cells (CD4, CD11b, CD11c, CD19, CD44, CD45R/B220, CD49b, TCRγ/δ, TER119) and streptavidin-coated magnetic particles. Labeled cells are separated using an EasySep‱ magnet without the use of columns.