Aqua live dead stain kit

PBMC were washed, stained (20 min; 4°C) for dead cells using the Aqua LIVE/DEAD stain kit (Invitrogen). Cells were then washed and stained (30 min; 4°C) with the following mAbs in different combinations: CD3, CD8, PD-1, 2B4, CD160 and CD57 or CD3, CD8, PD-1, 2B4, CD160, CCR7 and CD45RA. Cells were then permeabilized (1 h; 4°C) (Foxp3 Fixation/Permeabilization Kit; eBioscience) and stained (30 min; 4°C) with mAbs to EOMES and T-bet.

PBMC were stimulated overnight in complete media (RPMI (Invitrogen), 10% fetal calf serum (FCS; Invitrogen), 100 µg/ml penicillin, 100 unit/ml streptomycin (BioConcept)) with Staphyloccocus enterotoxin B (SEB; 250 ng/mL) or left unstimulated (negative control) in the presence of Golgiplug (1 µl/ml; BD) and anti-PD1, anti-CD160 and anti-2B4 mAbs. At the end of the stimulation period, cells were washed, stained (20 min; 4°C) for dead cells using the Aqua LIVE/DEAD stain kit (Invitrogen), permeabilized (20 min; 20°C) (Cytofix/Cytoperm, BD) and stained (30 min; 20°C) with mAbs to CD3, CD8, CD45RA, CCR7, IFN-γ and IL-2 [42] (link).

Peripheral blood mononuclear cells (PBMCs) were stimulated overnight in complete medium (RPMI [Invitrogen], 10% fetal calf serum [FCS; Invitrogen], 100 μg/ml of penicillin, 100 U/ml of streptomycin [BioConcept]) with ESAT-6 and CFP-10 peptide pools (1 μg/ml) or with Staphylococcus enterotoxin B (SEB; 250 ng/ml) or with HAd5 hexon-derived overlapping peptide pool, or they were left unstimulated in the presence of Golgiplug (1 μl/ml; BD) as previously described (53 (link)). At the end of the stimulation period, cells were washed and stained (20 min at 4°C) for dead cells using the aqua LIVE/DEAD stain kit (Invitrogen), washed, and stained (20 min at 4°C) with MAbs to CD3, CD4, CD8, PD-1, CCR7, and CD45RA. Cells were then permeabilized (30 min at 20°C) (Cytofix/Cytoperm; BD) and stained (20 min at 20°C) with MAbs to TNF-α, IFN-γ, IL-2, IL-4, IL-5, and IL-13. The criterion for scoring antigen-specific CD4 or CD8 T-cell responses as positive was as follows: the cytokine (IFN-γ, TNF-α, or IL-2) frequency obtained in the sample had to exceed 0.03% after background subtraction.

Cryo-preserved blood mononuclear cells (1–2×10⁶) cells were washed, stained (30 min; 4°C) for dead cells using the Aqua LIVE/DEAD stain kit (Invitrogen) and, when required, stained with appropriately tittered peptide-MHC class I multimer complexes at 4°C for 30′ in Ca2⁺-free media as described [24] (link). Cells were then washed and stained (30 min; 4°C) with the following mAbs: CD3, CD8, CD4, PD-1, CD160 and 2B4.

PBMC were washed, stained (20 min; 4°C) for dead cells using the Aqua LIVE/DEAD stain kit (Invitrogen). Cells were then washed and stained (30 min; 4°C) with the following mAbs: CD3, CD8, PD-1, 2B4, CD160, CCR7 and CD45RA. Cells were then permeabilized (20 min; 20°C) (Cytofix/Cytoperm, BD) and stained (30 min; 20°C) with mAbs to perforin and granzyme B [23] (link).

Overnight-rested cryopreserved blood mononuclear cells isolated from one HIV-1-infected elite controller individual (#1010) were stained with 0.25 μM 5,6-carboxyfluorescein succinimidyl ester (CFSE, Molecular Probes, USA) as previously described^{49 (link)}. Cells were then exposed to RV-Empty, RV-Gag, NYVAC-Empty or NYVAC-GPN vectors at various vector concentrations (0.1, 1 and 10 pfu/cell). In addition, cells were stimulated with GAG peptide (GPNHKARVL; internal control), 200 ng/ml of SEB (positive control; Sigma-Aldrich) or left unstimulated (negative control). At day 6, cells were harvested and stained (4 °C; 20 min) using the aqua LIVE/DEAD stain kit (Invitrogen) and Abs (4 °C; 30 min) to CD3, CD4, and CD8. Cells were fixed with CellFix (BD), acquired on an LSRII SORP (4 lasers: 405, 488, 532 and 633 nm) and frequencies of proliferating CFSE^low CD8 T cells were assessed using FlowJo (version 8.8.2; Tree star Inc, Ashland, OR, USA).