Anti cd8 pacific orange

Single cell suspensions of leukocytes from blood, ascites, tumor or omental metastases were stained with the following monoclonal antibodies: anti-CD3 APC-H7 (BD Pharmingen, 560176), anti-CD4 eFlour 450 (eBioscience, 48-0048-42), anti-CD8 Pacific Orange (Invitrogen, MHCD0830), anti-CD25 APC (BD Pharmingen, 555434), or anti-CTLA-4 APC (BD Pharmingen, 555855), anti-CD28 PerCpCy5.5 (eBioscience, 45-0289-42), anti-CD38 PE-TR (Invitrogen, MHCD3817), anti-HLA-DR PE-Cy7 (BD Biosciences, 335795), anti-FoxP3 PE (eBioscience, 12-4777-42), anti-ki67 FITC (BD Pharmingen, 556026) or CD39 FITC (BD Pharmingen, 561444) or anti-Helios (Biolegend, 137214). Cells were stained in FACs buffer (1%FBS in PBS with 0.01%NaN₃) and fixed according to the ebioscience FoxP3 Fix-Perm kit protocol (eBioscience, 00-5521-00). All samples were run on a BD LSRII Flow cytometer and analysed by FACSDiva BD. Briefly, for every single flow cytometric antibody, we have used Fluorescent Minus One (FMO), to discriminate between positive and negative cells [35 (link)].

Flow cytometry was performed as described previously [27] (link). Briefly, antigen-specific phenotypes and cytokine secretion profiles were assessed using a qualified polychromatic flow cytometry (PFC) panel. PBMC were co-incubated with peptide pools matched to the GRIN/ENV insert, 1 µg/ml SEB (Sigma-Aldrich, St. Louis, MO, USA) or mock stimuli, CD107a PECy5, BD Golgistop (Becton Dickinson, San Jose, CA, USA) and Brefeldin A (Sigma-Aldrich, Poole Dorset, UK) for 6 hours at 37°C. Cells were stained for viability with LIVE/DEAD® Fixable Violet Dead Cell Stain Kit (Invitrogen, Eugene, OR, USA), and then surface stained by anti-CD4 QD605, anti-CD8 pacific orange, anti-CD19 pacific blue (Invitrogen, Paisley, UK), anti-CD27 APC-H7, anti-CD14 pacific blue, anti-CD57 FITC, anti-B7 integrin PE (Becton Dickinson, San Jose, CA), and anti-CD45RO ECD (Beckman Coulter, High Wycombe, UK). Finally cells were stained intracellularly with anti-CD3 QD655 (Invitrogen, Paisley, UK), anti-IFNγ PE Cy7, anti-TNF-α A700 and anti-IL-2 APC (Becton Dickinson, San Jose, CA, USA) washed and acquired on the same day. At least 750,000 events were acquired on a custom-built BD LSR II cytometer. Data were analyzed and presented using FlowJo (version 8.8 Treestar).