2.7 Splenocytes in mice and Peripheral blood mononuclear cells (PBMCs) in cattle isolation
Spleen cells from immunized mice were removed at 120 dpv, stimulatedin vitro with iBoHV-1, and evaluated for antigen-specific proliferation. Splenocytes were obtained by gentle homogenization of spleens in supplemented RPMI 1640 medium/10% fetal calf serum (FCS, GIBCO)/1% ampicillin-streptomycin and gentamycin. Cells were counted using Turk.
Cattle blood samples were collected by venipuncture in syringes containing sodium citrate as an anticoagulant. PBMCs were isolated by centrifugation on Ficoll-Paqueâ„¢ PLUS (density 1.077 g/mL; GE Healthcare Bio-Sciences AB) as described elsewhere (Romera et al., 2000). Cells were counted using Trypan blue.