Abstract: The present invention relates to the isolation of human stem cells for use e.g. in cell therapy. More specifically, the invention is a method of separating mononuclear cells from human blood, which method comprises providing a human blood sample together with density gradient media (DGM) in a container; spinning the container comprising blood and DGM; and collecting the DGM fraction that comprises mononuclear cells; wherein the DGM has a density which is >1.080 and <1.090 g/cm3 as measured at 25° C. The invention also relates to bags and tubes which contain density gradient media wherein the density and osmolality has been optimized for use in the present method, preferably as kits.