2.10 Lymphoproliferation assay
Murine splenocytes were labeled with 3 μM carboxyfluorescein diacetate succinimidyl ester (CFSE) in PBS for 30 min at 37°C. Labeled cells were added to 96-well plates (5x105 cell/well) 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 at 37°C in 5% CO2 atmosphere for 4 days and then fixed with 0.2% paraformaldehyde. Cell proliferation was analyzed by flow cytometry using FACSCalibur® (Becton Dickinson, San Jose, CA) and Flowing Software (Turku Centre for Biotechnology, Finland). Results were expressed as delta proliferation and were calculated as the difference between the percentage of proliferating cells stimulated with inactivated FMD virus and the percentage of proliferating cells without stimuli.