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).