2.11 Surface and intracytoplasmatic staining for the detection of IFN-γ-producing murine splenocytes
Murine splenocytes were incubated in complete RPMI 1640 medium supplemented with 10% FCS and 50 mM 2-mercaptoethanol and were subjected to: (i) no stimulation ( mock), (ii) 2.5 μg/ml iFMDV or (iii) 5 μg/ml Concanavalin A (Sigma Aldrich®, St. Louis, MO) as positive control. Cells were incubated for 18 h in the presence of brefeldin A (BD GolgiPlug™), according to the manufacturer’s recommendations. After washing, cells were fixed in 0.5% paraformaldehyde and permeated with saponin (0.1% in PBS). Permeated cells were incubated for 20 min at RT with allophycocyanin (APC) anti-mouse INF-γ (clone XMG1.2, BD Pharmingen®) or isotype-matched control Abs. After 20 min, cells were washed twice and stained for 30 min at 4 oC with fluorescein isothiocyanate (FITC) anti-mouse CD4 (clone GK1.5, BDBioscience®); or phycoerythrin (PE) anti-mouse CD8 (clone 53-6.7, eBioscience®). Cells were then washed and fixed with 0.2% paraformaldehyde. Flow cytometry was performed as in 2.10 (Supplementary figure 1).