2.6 ALDCs Stimulation
Afferent lymph dendritic cells (ALDCs) from bovines were obtained by cannulation of pseudo afferent lymph vessels and characterized as previously described (Hope et al., 2006). Dendritic cells were incubated with culture medium RPMI 10% FBS (mock) or with 1 μg/mL of vaccines pCIgD, pCIgD-Man-L or plasmid free α1,2-Mannobiose-PEG2kDa-DOPE coated liposome (Man-L) with (+) or without (-) LPS from Brucella ovis . After 16 h incubation, a direct surface staining of cells was performed using monoclonal antibody DEC205-FITC (SEROTEC, UK), and an indirect surface staining was performed using monoclonal antibody anti CD40 (SEROTEC, UK), and anti-mouse IgG PE conjugated (Jackson laboratories, USA). Cells were fixed with 0.2% paraformaldehyde and acquired using FACScalibur cytometer and CellQuest software (BD).