Cxcr4 percpcy5

For multicolor flow cytofluorimetric analysis, PBMCs, LMNCs and PMNCs were stained with the following conjugated mAb as previously described: CD56-PE-Cy5 and NKp46-PE (Beckman Coulter, clone BAB281), CD16-PE-Cy7, CD19-APCH7, IFN-γ-PE, DNAM-1 PE, CD49e-PE, CD11c-PE, CCR5-PE, CD69-PE, CCR4-PE and CXCR3-PE (BD-Pharmigen), CD45-PB, CD62L-PE-Cy7, CCL5-AF647, CD161-PerCpCy5.5, CCR3-PE, CXCR2-PE and CXCR4-PerCpCy5.5 (Biolegend), CCR7-FITC and CXCR6-PE (R&D System), CX₃CR1 PE (MBL). Aqua LIVE/DEAD (Life Technologies) was used to eliminate dead cells from the analysis. Intracellular staining was performed using Cytofix/Cytoperm (Beckton Dickinson), according to the manufacturers instructions. The gating strategy used to select NK cells from both PBMC and LMNC is depicted in Supplemental Figure 1. For measurement of IFN-γ production, whole PBMC and LMNC were stimulated with 18 hours with 20ng/ml rhIL-12 (R&D Systems) and 200U/ml rhIL-2 (Peprotech). For the final 4 hours, GolgiStop was added (Beckton Dickenson). Flow cytometry data was acquired using an LSR Fortessa (Beckton Dickinson) and data was analyzed using FlowJo Software (Tree Star).