Cd158b pe

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 cytometric analysis was performed using cryopreserved PBMC. Viability was ascertained by trypan blue exclusion. Cells were washed, and then stained on ice for 20 min with the following fluorescent Abs in various combinations: CD3 (PerCP-Cy5.5 or allophycocyanin) or FITC, anti–TCR-αβ-1-FITC, and anti–TCR-γδ-1-PE (all BD Oncomark); Vα24 TCR-FITC and Vβ11 TCR-PE (Immunotech); CD56-PE, CD107a-PE, CD94-allophycocyanin, CD158b-PE, and CD16–PerCP-Cy5.5 (all from BD Pharmingen); and CD158a–PerCP-Cy5.5 (eBiosciences). After washing, stained cells were fixed in PBS/2% FCS/1.6% paraformaldehyde and acquired on a FACSCalibur flow cytometer (BD Biosciences). For intracellular staining, cells were first surface stained, followed by washing and incubation for 30 min on ice with Fix/Perm buffer (eBioscience). After washing in permeabilization buffer, cells were incubated for 30 min on ice with perforin-PE (Perforin reagent set; BD Pharmingen 556437) or IFN-γ allophycocyanin. The cells were washed again, fixed in PBS/2% FCS/1.6% paraformaldehyde, and acquired. Data were analyzed using Flowjo software (Tree Star, Ashland, OR).