Panotic

Briefly, mice were submitted to euthanasia 24 h after surgery using isoflurane (Cristália). The peritoneal cavity was washed with 3 mL of cold sterile saline in the laminar flow cabinet. The peritoneal washes were plated in Difco tryptic soy agar (TSA) (BD) for further analysis of bacterial growth through the count of CFU.
The peritoneal washes were also used for total cell count. Red blood cells were lysed using Turk solution (2% acetic acid) and total cell count was carried out using Neubauer chamber (Neubauer Improved). Differential leukocyte count was performed in cytocentrifuged smears stained with panotic (Laborclin). The supernatant was collected by centrifugation and stored at −20°C for further cytokine quantification.

BMMΦ were infected with 2 × 10⁶ promastigotes of either R0 or R60, grown for 7 days (late stationary phase) in axenic cultures. The parasites were centrifuged at 50 g for 10 min; dead parasites were discarded, and the viable ones were added to the BMMΦ culture in an average proportion of five parasites per BMMΦ. After 2 h of homogenization, the cells were washed with sterile PBS to eliminate the non-internalized parasites (time 0 h). The wells were analyzed after 3, 12, and 24 h (times 3, 12, and 24 h, respectively).
To evaluate in vitro infectivity, 8 × 10⁵ cells/mL were plated in chamber slides (Lab-tek chamber slides, NUNC, Thermo Fisher Scientific). Panotic (Laborclin, Pinhais, PR, Brazil) was used to stain slides, following manufacturer instructions. Olympus BX50 optical microscopes (Olympus, Center Valley, PA, USA) were used for microscopic examination of slides to determine BMMΦ infectivity. A minimum of 300 BMMΦ per chamber well was analyzed for each SIVP cultures and for each time interval, and the numbers of infected and non-infected cells were determined. The experiment was performed in triplicate. Two-way ANOVA was used for statistical analyses.

500μL of blood was collected by cardiac puncture and transferred to tubes containing the anticoagulant EDTA (Vacuplast, Brazil). Global leukocyte counts were performed using a Bio-2900 Vet automated hematology counter. Blood smears were stained with Panotic (Laborclin, Brazil) for differential leukocyte counts, and 100 leukocytes were differentiated under a light microscope.