The unlabeled cells ran through, thus this cell fraction was depleted of CD4+ or CD8+ cells. After removal of the column from the magnetic field, the magnetically retained CD4+ or CD8+ cells were eluted as the positively selected cell fraction by washing the magnetic CH5424802 datasheet column with 15 mL of isolation buffer. The purity of CD4+ and CD8+ T cells was evaluated by flow cytometry on a FACSCalibur instrument (Becton Dickinson, USA) interfaced to an Apple G3 workstation. Cell-Quest software (Becton Dickinson, USA) was used for both data acquisition and analysis. A total of 20,000 events were

acquired for each preparation. Flow cytometric analysis was performed using canine whole blood leukocytes that were selected on the basis of their characteristic forward (FSC) and side (SSC) light-scatter distributions. Following FSC and SSC gain adjustments, the lymphocytes were selected by gating on the FSC versus SSC

graph. Fluorescence was evaluated from FITC spectra (anti-CD4 and anti-CD8 antibodies) on FL1 in dot plot representations. A marker was set as an internal control for nonspecific 3-Methyladenine in vitro binding in order to encompass >98% of unlabeled cells, and this marker was then used to analyze data for individual animals. The results are expressed as the percentage of positive cells within the selected gate for cell surface markers presenting CD4 or CD8. Statistical analysis was performed using instrumental support of the software GraphPad Prism 5.0 (Prism Rebamipide Software, USA). Data normality

was demonstrated by the Kolmogorov–Smirnoff test. The analyses of the macrophage cultures (% of infection and number of amastigotes), NAG, and MPO were performed by ANOVA employing repeated measures (paired). Data were considered statistically significant when the p value was <0.05. During the cultivation period, changes were observed in cultures of monocytes adhered to cover slips that differentiated into macrophages, 2, 3, 4, and 5 days after culture began (Fig. 1). At 2 and 3 days of differentiation, even after wells were washed, large numbers of granulocytes as well as mononuclear cells remained attached (Fig. 1A and B). In contrast, monocytes differentiated into macrophages by the fourth day of culture already demonstrated morphological changes such as increased size, cytoplasm vacuolation, and irregular shape (Fig. 1C). On the fifth day of maturation, these morphological changes remained, and there was an increase in cell size, number of nuclei (giant cells), and cytoplasm vacuolation (Fig. 1D). The phagocytic ability of monocytes that had differentiated into macrophages was assessed 3 h after L. chagasi promastigotes were used to infect monocytes at 2–5 days of differentiation ( Fig. 2). These monocytes were then analyzed 24, 48, 72, and 96 h after L. chagasi infection. As shown in Fig. 2A, the percentage of macrophages infected by L. chagasi was statistically higher (p < 0.05) based on the length of time monocytes had differentiated into macrophages.