Isolation of Canine Peripheral Blood Mononuclear Cells

Blood samples (10 ml each) from the 17 dogs (control group: n = 6; Leishmune® group: n = 5; LdCen^−/− group: n = 6) were collected in heparinized tubes intended for obtaining peripheral blood mononuclear cells (PBMC), as previously described [13 (link)]. Briefly, the whole blood volume collected was placed in a mixture of Ficoll-Hypaque (Sigma Chemical Co.; density: 1.119 g/ml) and Ficoll-Hypaque (Sigma Chemical Co.; density: 1.077 g/ml) at a 1:3 ratio (Ficoll/blood) in sterile polystyrene conical bottom tubes (Falcon, Corning, USA). All samples were centrifuged at 700 × g for 80 min at 22 °C. The PBMC ring was collected at the Ficoll-Hypaque interface and transferred to another tube with 40 ml of Falcon sterile 1× PBS containing 10 % FBS. This tube was centrifuged twice at 400 × g for 10 min at 4 °C. After the supernatant was discarded, the cells were resuspended in 1 ml of cell culture medium RPMI 1640. Cells were counted in a Neubauer hemocytometer chamber to determine the numbers of monocytes or lymphocytes per milliliter.