01% sodium azide (fluorescence-activated cell sorting [FACS] wash), and fixed in 200 μL of 2% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA). Isotype-matched fluorescently labeled control antibodies were used to determine background levels of staining. Lymphocytes were identified by characteristic forward and side scatter
parameters, and populations of interest were gated on patterns of CD56/CD3 staining within the lymphocyte population. Results are expressed as the percent positive of the gated population. Intracellular perforin staining was performed after permeabilization with 0.2% saponin using a δ-G9 antibody obtained from BD Biosciences. Thawed mononuclear cell suspensions were enriched for NKs using the NK Isolation Kit II from Miltenyi NSC 683864 in vivo Biotec (Gladbach, Germany) according to the manufacturer’s instructions. The median purity of NKs was >90% in all cases. After isolation, NKs were cultured with or without IL-2 (25 ng/mL, R&D Systems) for
48 hours at 37°C and 5% CO2. Following culture, carboxyfluorescein succinimidyl ester–labeled target cells (K562s) were added to NKs at effector-to-target FK506 ic50 concentrations of 0:1 (negative control) and 10:1 (test) and incubated at 37°C for 4 hours. After incubation, cytotoxicity was measured using the flow cytometry–based Total Cytotoxicity & Apoptosis Detection Kit from Immunochemistry (Bloomington, MN). Immediately before acquisition, 7-aminoactinomycin D was added to effector-to-target populations and incubated for 15 minutes on ice. Cells treated with 0.1% Triton-X below served as positive controls. Degranulation was determined by way of flow cytometric analysis of increased CD107a (Lamp, BD Bioscences) expression on bead-isolated
NKs after 4-hour stimulation with phorbol myristate acetate (PMA) (10 ng/mL) and ionomycin (1 μg/mL) in the presence of brefeldin A (Sigma-Aldrich) and CD107a. NKs cultured under the same conditions without PMA and ionomycin served as unstimulated controls. Antibodies for intracellular interferon-γ (IFN-γ) were supplied by BD Biosciences. Thawed mononuclear cells were stimulated with PMA (10 ng/mL) and ionomycin (1 μg/mL) for 4 hours at 37°C in the presence of brefeldin A. After stimulation, cells were stained for surface antigens (as above), fixed for 30 minutes at 4°C in 100 μL Fix and Perm Medium A (Caltag, Burlingame, CA), permeabilized using 100 μL Fix and Perm Medium B (Caltag), and incubated with anti-cytokine monoclonal antibody for 1 hour at 4°C in the dark. Cell suspensions were then washed in FACS wash and fixed in 200 μL 2% paraformaldehyde and acquired after 1 hour. Cells cultured under the same conditions in the absence of PMA and ionomycin served as controls. NKs were enriched using magnetic beads and were surface-stained for CD3, CD56, and NKp30 as described above.
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