Spleens were harvested into single cell suspensions by pulverization through a 70 micron filter as previously described (35 (link)). After lysis of red blood cells, cell suspensions were washed in pre-warmed (RT) HBSS/10% FBS and loaded onto a nylon wool column prepared in a 10 ml syringe. Cells were incubated on the column at 37°C for 1 hr. Non-adherent cells were passed through the column after the addition HBSS/10% FBS, collected and grown in T-75 flasks (BD Biosciences) in the presence of IL-2 (8000 U/mL; Proleukin, Chiron). After 4 days, non-adherent cells were removed and flasks were washed with PBS to remove loosely adherent cells. Fresh IL-2 containing media was replenished in the original flasks. Washes were collected with the non-adherent cells, centrifuged and transferred to a second flask with IL-2 containing media. Cells were maintained for an additional 3–4 days before use. This protocol yielded LAK cells in which 60–90% of the cells were NK1.1+, CD3−.