Methionine free media

NK cells were isolated from peripheral blood using the Human NK Cell Isolation Kit and the autoMACS instrument (Miltenyi Biotec) according to the manufacturer's instructions. NK cell purity was around 100% as determined by FACS. The NK cells were grown in the presence of IL-2 and the cytotoxic activity of NK cells against various targets was assessed in 5hr ³⁵S release assays as described [39 (link)]. Briefly, target cells were grown over night in the presence of ³⁵S- methionine added to a methionine-free media (Sigma). For each target, the spontaneous ³⁵S release was measured from the supernatants of target cells which were not incubated with effector cells. Maximum ³⁵S release was calculated by adding 0.1M of NaOH to the target cells. Following 5 hour incubation with effector cells, the level of ³⁵S release was measured by a MicroBeta2 Plate Counter (Perkin Elmer). CD107a mobilization assays were performed as described [40 (link)]. CD107a surface expression was determined by FACS and the maximum percent of positive cells (in the mock population) was set to be 100%.

NK cells were isolated from healthy donors via MACS separation kit (Miltenyibiotech) and grown in the presence of IL-2 (peprotech). Target cells were grown over night in the presence of ³⁵S added to a Methionine-free media (Sigma). Prior to incubation with the effectors, cells were washed, counted, and 5000 cells/well were plated. For each target, the spontaneous ³⁵S release was calculated by cells which were not incubated with effector cells, and maximum ³⁵S release was calculated by applying 0.1M of NaOH to the target cells. The level of ³⁵S release was measured after 5 hours of incubation with effectors (at 37°_c) by a β-counter TopCount (Packard). When blocking antibody was used, NK cells were preincubated with 0.5 ug/well of the blocking antibody on ice for 1 hour, and then the ³⁵S labeled target cells were added for 5-hour incubation.