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).