Isolation of Natural Killer Cells from Mouse Bone Marrow and Spleen

Abstract

Natural killer cells are large granular lymphocytes that are particularly important for killing virus infected cells and tumor transformed cells. Unlike T cells, NK cells are unique in their ability to kill target cells without prior sensitization. Hence, NK cells are very effective mediators for immune-modulation in combinatorial immunotherapy against tumors. The present chapter describes isolation of NK cells from spleen and bone marrow of mice and their purification. The chapter outlines a protocol for purification of NK cells isolated from spleen and bone marrow by depletion of contaminating cells in the preparation using negative selection process. Monoclonal antibody cocktail and magnetic separation is used to deplete contaminating cells. This process of negative selection has the advantage of the NK cells (cells being enriched for) not being coated with antibodies and hence not at risk of functional activation by antibody cross-linking and also helps in selecting all NK cells and not differential isolation of any skewed NK population. NK cells thus purified, have also been assessed for purity using flow cytometry by use of fluorescent tagged monoclonal antibodies for pan-NK markers. The functional potential of the NK cells isolated and purified have also been evaluated using cytotoxicity assay against prototypic tumor target cells.