Complete blood counts (CBCs) were performed at the time of sample collection, and the results were subsequently used to calculate the absolute number of NK cells following flow cytometric analysis. Ethical approval was obtained
from the Federal University of São Paulo IRB, and patients gave informed consent. Cryopreserved peripheral blood mononuclear cells (PBMCs) were thawed and used for measurements of NK cell frequency, number and receptor expression. The thawed cells were washed with RPMI-1640 medium supplemented with 15% fetal bovine serum (FBS) before staining or stimulation. NK cell function was assessed by cytokine flow cytometry (CFC). To measure NK cell function, PBMCs were cultured in medium alone, or stimulated with K-562 cells (10 : 1 effector Dasatinib solubility dmso to target ratio). The PBMCs cultured in medium alone were taken as a measure of ‘spontaneous’ NK cell function. Briefly, 100 μl of thawed PBMCs was stimulated at 5 × 106 cells/ml in 96-well plates (5·0 × 105/well) in the presence of 10 μg/ml fluorescein isothiocyanate (FITC)-conjugated anti-CD107a antibody for 24 hr; during the last 6 hr of culture, monensin and brefeldin-A were added to block trans-Golgi transport and allow intracellular accumulation of cytokines. The cells were then harvested,
washed in buffer and prepared for antibody staining and Staurosporine ic50 flow cytometry. Cryopreserved specimens were used for measurements of NK cell frequency, number and receptor expression. The thawed cells were washed with phosphate-buffered saline (PBS) supplemented with 1% bovine serum albumin (BSA) and 2 mm ethylenediaminetetraacetic acid (EDTA) [fluorescence-activated acetylcholine cell sorting (FACS) buffer] before staining. For staining, 5 × 105 cells were incubated with purified human immunoglobulin G (IgG; 100 μg/ml) to block non-specific binding. For the gating strategy, doublets were excluded based on forward scatter (FSC) height and
FSC area (Fig. 1a). A broad PBMC gate was then used based on FSC height and side light scatter (SSC). Monocytes, B cells and T cells were excluded based on CD14, CD19 and CD3 gating, respectively (Fig. 1a). NK cells were gated from the CD14-, CD19-, CD3-negative lymphocyte population and were then subdivided into CD56bright, CD56dim and CD56neg populations and analysed for the expression of the NK cell activating receptors NKp30 and NKp46, and for CD107 expression. We used commercially available anti-KIR antibodies DX9 and Z27 to further phenotype the NK cells (BD Biosciences, San Jose, CA). Fluorescence minus one (FMO) samples were prepared for each fluorochrome to facilitate gating. All cells were analysed by flow cytometry using a two-laser FACSCanto instrument running facs-diva software (BD Biosciences). Anti-mouse IgG-coated beads (BD Biosciences) were stained with each fluorochrome separately and used for software-based compensation.