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).