Fix and perm cell fixation and cell permeabilization kit

Th1 differentiation was assessed by co-culture of sorted naive CD4⁺ T cells and infected B cells 5 dpi. 1 × 10⁵ naive CD4⁺ T cells stained with CellTrace Violet (Thermo Fischer Scientific) and 0.5 or 1 × 10⁵ infected B cells were cultured in 96-well plates with Dynabeads Human T-Activator CD3/CD28 (Thermo Fischer Scientific) and cultivated for 7 d. The neutralizing antibody against IL12B (C8.6; BioLegend) or the corresponding isotype control antibody (MOPC-21; BioLegend) were added for certain experiments at 5 µg/ml. Cells were restimulated with PMA and ionomycin (Cell Stimulation Cocktail; eBioscience) for 5 h and treated with Brefeldin A and Monensin (BioLegend) for 2.5 h before fixation. Th1 population was measured by intracellular IFN-γ staining with FIX and PERM Cell Fixation and Cell Permeabilization kit (Thermo Fischer Scientific) and subsequent flow cytometry analysis. The Th1 population was defined as IFN-γ⁺ T cells in the fraction of proliferating T cells identified via CellTrace Violet staining. EBV-specific effector T cells’ activities were measured with ELISA and Calcein release assays. For IFN-γ detection from T cells, effector and target cells were seeded at 5 × 10⁴ cell per ml (1:1 ratio) each and co-cultured for 16 h in a 96-well plate (V bottom). IFN-γ levels were detected with ELISA. IFN-γ concentrations <16 pg/ml were considered as not detected.