Anti vδ2 fitc

The expanded γδ T cells (2 × 10⁵) were pelleted by centrifugation at 400 × g for 5 min, followed by resuspension in 40 µl of PBS. Two appropriate fluorochrome-labeled monoclonal antibodies (mAbs; each 5 µl) were added to stain the relevant surface markers. After incubation at 4 °C for 1 h, the stained cells were washed once with PBS and analyzed on a flow cytometer (#0500-5009, Guava easyCyte 5 HPL/guavaSoft software version 2.1, Millipore) where at least 10,000 events were acquired per sample. The collected data were analyzed with FlowJo software (version 10.0.8r1; Attached Dongle ID: AA04012700018517) and presented as dot plots with quadrant gates. The following mAbs were used: anti-Vγ9 FITC (#TCR1720, Invitrogen), anti-Vδ2 PE (#555739, BD), anti-Vδ2 FITC (#562088, BD), anti-CD3 FITC (#555332, BD), anti-CD16 PE (#555407, BD), anti-CD27 PE (#560985, BD), anti-CD45RA FITC (#561882, BD), anti-NKG2D PE (#561815, BD), and anti-PD1 PE (#560908, BD).

The following monoclonal antibodies were used to characterize MoDC: anti-CD86 FITC, anti-CD1a PE, anti-HLA-DR PERCP, anti-CD83 APC, anti-CD14 APC-H7, anti-CD80 FITC, anti-CD40 PE, anti-HLA-I APC, anti-CCR7 PE-Cy7, anti-CCR5 APC-H7 from BD Biosciences, and anti-BT3A.1 PE from BioLegend. To evaluate γδ T lymphocytes phenotype we used anti-Vδ2 FITC, anti-CD3 PE, anti-CD69 PERCP, anti-CD45RA PE-Cy7, anti-CD27 APC, anti-CD16 PACIFIC BLU, anti-CD25 APC-H7 (BD Biosciences). In brief, the cells were washed twice in PBS, 1% BSA, and 0.1% sodium azide, and were stained with the mAbs for 15 min at 4°C. The cells were then washed and fixed with 4% paraformaldehyde, and analyzed using a FACS Canto II (Becton Dickinson). Since the presence of 2 purified populations, the gating strategy was performed as follow: dead cells were excluded by scatter characteristics; MoDC were identified by morphological parameters (FSC vs SSC); gated cells were then analyzed for the expression of the molecules described above. T lymphocytes were first gated by using morphological parameters; in this gate Vγ9Vδ2 T cells were identified as Vδ2+CD3+. Analysis was carried out by using Facs Diva software (Becton Dickinson). The histogram overlays were performed by FlowJo software (TreStar, Olten, Switxìzerland).