Canine DCs were prepared from peripheral blood (PB) monocytes, as described previously
[8 (link)]. PB monocytes were isolated by magnetic cell
sorting using anti-hCD14 microbeads (Miltenyi Biotec GmbH, Bergisch-Gladbach, Germany). To
induce their differentiation into DCs, the isolated PB monocytes were incubated with
rcGM-CSF (R&D Systems Inc., Minneapolis, MN, USA) and rcIL-4 (R&D
Systems) for seven days. More than 95% of the resulting cells showed high expression of
CD40 and CD80, which are co-stimulatory molecules highly expressed on DCs.
As previously described [21 (link)], the T cell fraction
was prepared by incubating PBMCs in a nylon wool column (Wako Pure Chemical Co., Ltd.,
Osaka, Japan). Seventy-eight percent of the T cell fraction expressed CD3. Moreover, the
population did not express CD14, a marker of monocytes, or CD21, a marker of B cells.
[8 (link)]. PB monocytes were isolated by magnetic cell
sorting using anti-hCD14 microbeads (Miltenyi Biotec GmbH, Bergisch-Gladbach, Germany). To
induce their differentiation into DCs, the isolated PB monocytes were incubated with
rcGM-CSF (R&D Systems Inc., Minneapolis, MN, USA) and rcIL-4 (R&D
Systems) for seven days. More than 95% of the resulting cells showed high expression of
CD40 and CD80, which are co-stimulatory molecules highly expressed on DCs.
As previously described [21 (link)], the T cell fraction
was prepared by incubating PBMCs in a nylon wool column (Wako Pure Chemical Co., Ltd.,
Osaka, Japan). Seventy-eight percent of the T cell fraction expressed CD3. Moreover, the
population did not express CD14, a marker of monocytes, or CD21, a marker of B cells.