2.8 Lymphoproliferation Assay
Splenocytes and PBMCs suspensions obtained from each animal were labeled with 3 µM carboxyfluorescein diacetate succinimidyl ester (CFSE) for 30 min at 37 °C. Cells were washed and resuspended in RPMI 1640 complete medium (RPMI 1640 10% FBS, 10 mM HEPES, 100 U/ml penicillin, 100 mg/mL streptomycin, and 50 mM 2-mercaptoethanol). CFSE-labeled PBMCs or splenocytes were added to a 96-well plate (U-bottom) at a concentration of 5x105 cells per well, in 100 µL of complete medium containing moi 1 of inactivated BoHV-1, concanavalin A (ConA) (Sigma-Aldrich, St. Louis, MO, USA), or medium as a positive or negative proliferation control, respectively. Cells were maintained at 37 °C in 5% CO2 atmosphere. After 4 days incubation of cells from mice or 5 days for cells from cattle, cells were fixed with 0.2% paraformaldehyde. Cell proliferation was analyzed by flow cytometry, using a FACSCalibur (Becton Dickinson, San Jose, CA, USA) and CellQuest software (Becton Dickinson).