Pe anti mouse tnf α

Fresh mouse splenocytes were plated in a 96 round-bottom well plate (Costar, Corning Inc., Corning, NY, USA) at a density of 2 × 10⁶ cells/well and incubated overnight with either 1 µM/peptide of the ZIKV E peptide pool, or 5 µM of YFV-17D NS3 ATLTYRML or 50 µg/ml of Vero E6 cell lysate. After treatment with 5 µg/ml brefeldin A (Biolegend, San Diego, CA, USA) for 2 h, the splenocytes were stained for viability with Zombie Aqua™ (Biolegend) (1:200 dilution in PBS) for 15 min. Subsequently, extracellular markers CD3 (4 µg/ml eFluor® 450 anti-mouse CD3 antibody, ThermoFisher, Waltham, MA, USA) and CD8 (2 µg/ml APC/Cy7 anti-mouse CD8a antibody, Biolegend) were stained and cells were fixed in 2% paraformaldehyde (Sigma-Aldrich, St. Louis, MO, USA). Finally, splenocytes were permeabilized with 0.1% saponin before intracellular staining for IFN-γ (2 µg/ml APC anti-mouse IFN-γ, Biolegend), TNF-α (6.5 µg/ml PE anti-mouse TNF-α, Biolegend) and granzyme B (5 µg/ml FITC anti-human/mouse granzyme B, Biolegend). Samples were analyzed on a BD LSRFortessa™ X-20 (Becton Dickinson, Franklin Lakes, NJ, USA). Percentages of responding CD4⁺ or CD8⁺ T cells were calculated by subtracting the percentage of responders from non-stimulated samples (incubated with non-infected Vero E6 cell lysates) from corresponding stimulated samples.