The cultures were collected, washed, incubated for 15 min with mouse mAbs against human CD3-PerCP, CD56-FITC, or PE, CD69-APC, CD16-PE (BD Biosciences, USA), and NKG2D-PE (BioLegend, USA). NK cells were incubated with CD158a-PE and CD158b-PE (BD Pharmingen, USA), CIK cells were incubated with CD4-PE and CD8-APC (BD Biosciences) and γδ T cells were incubated with Vγ9-FITC (BD Pharmingen), CD4-PE, and CD8-APC. Isotype-matched antibodies were used as controls. Perforin and granzyme B detection was performed according to the BD Cytofix/Cytoperm™ Kit manual (BD Biosciences). Briefly, NK, CIK, and γδ T cells were harvested and adjusted to 1 × 106 cells/mL in RPMI-1640 medium containing 10 % fetal calf serum, and incubated 0.1 % GolgiStop (BD Biosciences) for 4 h. After pre-incubation with 10 % normal human serum, cells were stained with mAbs to identify NK (CD3−CD56+), CIK (CD3+CD56+), and γδ T cells (CD3+Vγ9+), followed by intracellular staining for perforin-PE and granzyme B-PE (BD Pharmingen), and the corresponding isotype antibodies to determine intracellular cytokine levels.
Flow cytometry data acquisition was performed on a BD FACS Calibur (BD Biosciences) with Cell Quest Pro software. Analysis was performed with FlowJo software (Tree Star, USA).
Flow cytometry data acquisition was performed on a BD FACS Calibur (BD Biosciences) with Cell Quest Pro software. Analysis was performed with FlowJo software (Tree Star, USA).