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).
Free full text: Click here
