Diluent c

The CM of M-protein-treated and untreated BCC MDA-MB-231 cells was collected and centrifuged at 300×g for 5 min, followed by 1200×g for 20 min. The supernatant was ultracentrifuged at 140,000×g for 70 min at 4°C using an Optima XE-100 ultracentrifuge to collect the EV pellet. EV pellets were collected in 100 μL of PBS; these were considered to be isolated EV. The EV collected from the M-protein-treated BCC were called MpEV, and the EV collected from the untreated breast cancer cells were called nEV. For PKH26 labeling of EV, of 120 µg was resuspended in 250 µL of Diluent C (Sigma-Aldrich) and mixed with 250 µL of Diluent C containing 1 µL of PKH26 (Sigma-Aldrich). The mixture was then incubated in the dark for 5 min and neutralized by adding 40 ml of PBS containing 0.25% FBS. The solution was then ultracentrifuged at 140,000×g for 70 min at 4°C to collect stained EV pellet, then resuspended in 100 µL PBS and stored at -80°C. The protein concentration of EV was measured using a Bradford assay (Bio-Rad, Hercules, CA, USA) and was considered to be the EV concentration. The size of collected EV was measured by dynamic light scattering (Zetasizer Nano ZS, Melvern Instruments, United Kingdom). EV markers expression was characterized by Western Blotting.

After isolation or rapid thawing, the amastigotes to be labeled were washed in plain RPMI medium (Gibco, Invitrogen) and in DPBS (Mg²⁺ and Ca²⁺ free; Gibco, Invitrogen) before resuspension in 100 μL diluent C (Sigma) per 10⁸ amastigotes. An equivalent volume of diluent C containing 1:250 PKH67 membrane linker dye (Sigma) was added to the suspension and incubated for 3 min at room temperature. The staining reaction was stopped by adding an equivalent volume to the total amount of diluent C of FBS to the parasites and incubating at room temperature for 1 min. The thus-stained parasites were washed in plain RPMI medium and complete IMDM before use in subsequent experiments.