Inf γ

Cytokines in cell culture media were measured by ELISA as follows. Tumour cells were incubated with sEVs for 24 or 48 h, and INF-γ (BioLegend) levels were measured in the culture medium.
Levels of aspartate aminotransferase (Abcam), alanine aminotransferase (Abcam), blood urea nitrogen (Abcam), and creatinine (Thermo Fisher Scientific) in serum after 4 h for the systemic administration of sEVs were also tested using ELISA follow manufacturer’s protocol for the biosafety measurements.

The cytokine titration and the evaluation of the specific peripheral immunologic profile were performed as described previously [5 (link)]. Briefly, the pro- and anti-inflammatory human cytokines IL-1β, IL-4, IL-5, IL-6, IL-17A, CCL5, and INF-γ (all from BioLegend, San Diego, CA, USA) were determined with commercial sandwich enzyme linked immunosorbent assay (ELISA) kits, following the supplier’s instructions. For the specific immunologic profile, the PBMC cells stimulated with T. solium vesicular fluid or medium alone was measured using standard phenotyping protocols provided by the manufacturers. Cellular populations of naive cells (CCR7+/CD45RA+), central memory (CCR7+/CD45RA−), effector (CCR7−/CD45RA−/CD27+/CD28+), B cells (CD19+), T Regs (CD4+/CD25+/FoxP3+), and NK (CD3−/CD16+/CD56+) were determined.

Macrophages were seeded at 2.5 × 10⁵ cells per well in 24-well plates. After attachment to the wells, the cells were primed using interferon gamma (INFγ; carrier-free; 150 U/mL; Biolegend no. 57308) for 16 h prior to the bacterial infections. The macrophages were infected with stationary-phase salmonellae at a multiplicity of infection (MOI) of 10:1 by centrifugation for 10 min at 4°C. The infectious dose was corroborated by serial dilutions and plating, aiming to infect the macrophages with the same initial CFU. For all the experiments that involved infection of macrophages, the initial CFU was not statistically different between strains across the independent biological replicates. Infected macrophages were incubated at 37°C under 5% CO₂ for 1 h and washed three times with PBS to remove extracellular bacteria. 250 μL of RPMI + FBS with 100 μg/mL of gentamicin sulfate (VWR no. 0304) was added to the wells to be collected at 2 hpi, and 10 μg/mL of gentamicin was added to the wells to be collected at 6 hpi At the corresponding time, cells were washed with PBS, and PBS + 0.1% Triton X-100 was added for macrophage lysis, and monolayers were gently scraped and collected. Three wells per bacterial genotype were assessed per time point. Surviving intracellular CFU were enumerated by plating serial dilutions.