*4.7. Hemagglutinating and Antimicrobial Activities*

Human erythrocytes (type A) were collected in 0.15 M citrate buffer, pH 7.4, and washed 3 times by centrifugation with 0.15 M PBS, pH 7.4. To assess the hemolytic activity, aliquots of 10 μL of the selected toxin at 0.01, 0.1, 1, or 10 μg were put in 50 μL in a 3% suspension of erythrocytes in wells of U-shaped bottom plates and incubated for 3 h at room temperature. Solutions of isolated toxin at 10 μg previously incubated with D-Galactosamine (12662, Sigma) or D-Mannose (M2069, Sigma) both at 10 or 30 mM for 1 h were also usedThe hemagglutinating activity was verified by reading the absorbance at 595 nm of each well in a plate reader. Erythrocytes incubated with water were used as positive control (100% hemolysis). Antimicrobial potential was monitored by a liquid growth inhibition assay against *M. luteus* A270, *E. coli* SBS 363, and *C. albicans* MDM8, as described by Rossi et al. [56]. Pre-inoculum of the strains was prepared in Poor Broth (PB broth, 1.0 g peptone in 100 mL of H2O containing 86 mM NaCl at pH 7.4; 217 mOsM for *M. luteus* and *E. coli* and 1.2 g potato dextrose in 100 mL of H2O at pH 5.0; 79 mOsM for *C. albicans*), at 37 ◦C under agitation. The absorbance at 595 nm was set on, and one aliquot of this solution was taken to get cells in logarithmic growth (A595 nm ~0.6) and diluted 600 times (A595 nm = 0.0001). The isolated toxin or Nattectin, both at 10 μg, was dissolved in sterile Milli-Q water to 100 μL of PB broth. Tetracycline and Gomesin were used as inhibitor controls. After 18 h of incubation at 30 ◦C, the inhibition of bacterial growth was determined by measuring absorbance at 595 nm.

#### *4.8. In Vivo Experimental Protocol for Intravital Microscopy*

Initially, Balb/c mice were injected by the intrascrotal route with 50 μL of TmC4-47.2 at 10 μg and rested for 3 h. Independent groups of mice were pre-treated with 500 μL of intraperitoneal (i.p.) injection of the purified rat anti-mouse CD29 (beta 1 integrin, 14-0299-82, eBioscience), hamster anti-mouse CD49a (alpha 1 integrin, PA5-95563, Thermo Fisher eBioscience), rat anti-mouse CD49b (alpha 2 integrin, ab238665, Abcam), rat antimouse CD49e (alpha 5 integrin, ab221606, Abcam), and rat anti-mouse CD106—VCAM-1, 14-1061-82, Thermo Fisher eBioscience) monoclonal antibodies at 10 μg mL−1, 30 min before TmC4-47.2 injection. Control animals received the same amount of control isotype IgG. Negative control was injected with intrascrotal sterile PBS. The study of the microvascular system was performed with an optical microscope (Axio Imager A.1, Carl-Zeiss,

Oberkochen, DE ) coupled to a photographic camera (IcC 1, Carl-Zeiss, Germany) through a 10/025 longitudinal distance objective/numerical aperture and 1.6 optovar. The surgical cremaster preparation was handled as described previously [12]. Mice were anesthetized by an i.p. injection of 2% Xylazine—(Calmiun®, Agener União, São Paulo, Brazil) and with 0.5 g Kg−<sup>1</sup> of ketamine (Holliday-Scott SA, Buenos Aires, Argentina). The scrotum was exposed, and the cremaster muscle reached. Following the incision with cautery and spreading the muscle over a cover glass, the epididymis and testis were mobilized and pinned aside, allowing the microscopic access to the muscle microcirculation. The exposed tissue was superfused with 37 ◦C warmed bicarbonate-buffered salines, pH 7.4. The post-capillary venules with a diameter of 25–40 μm were chosen, and the interaction of leukocytes with the luminal surface of the venular endothelium was evaluated, counting the number of rolling leukocytes every 10 min after application of inflammatory agent for 30 min. Rolling leukocytes were defined as those moving at a velocity less than erythrocytes and demonstrated a clear rolling motion. The number of adherent cells was expressed as the number per 100 μm length of venule. The experiments were carried out under the National Council for Animal Experiment Control (CONCEA) and approved by the Butantan Institute's Animal Use Ethics Commission (CEUAIB #275/06).
