Intracellular Cytokine Assay
Intracellular staining to characterize T cell populations based on cytokine profile was developed. Cells were stimulated with 2μl/ml Cell Activation Cocktail (PMA/ionomycin, BioLegend, UK), for 2 hours, then brefeldin A (5μg/ml, BioLegend, UK) was added for a further 3 hours. Non-activated cells treated with brefeldin were used as negative controls. Cells were harvested, washed twice in PEA and surface markers labelled with CD8-Viogreen, CCR7-FITC, CD57-PE and CD45RO-PE-Vio700 (Miltenyi) as before, washed in PBS plus 2.5mM EDTA (PE buffer) and stained with 1μl/ml Fixable Viability Dye eFluor™ 780 (eBioscience, UK) for 30 minutes at 4°C. Cells were washed again in PE, then fixed and permeabilized as per manufacturer’s instructions (BD Biosciences, UK) and labelled with anti-IFN-γ-eFluor450 (eBioscience, Thermo UK), anti-TNF-α-PE-Vio770 and anti-IL-2-APC (both Miltenyi) for 30 minutes. Cells were washed again and resuspended in PEA and up to 100,000 live events recorded on MACSQuant10 / BD Fortessa LSR.