Earle s salts

Neospora caninum tachyzoites (NcT) from strain Nc-1 (ATCC™ 50,843) were isolated from infected VERO cell cultures as previously mentioned, with slight modifications^{28 (link),31 (link),32} . Briefly, VERO cells infected with NcT were cultured at 37 °C and 5% CO₂, in minimum essential medium with Earle’s salts (Corning, NY, Missouri, USA) supplemented with 2 mM L-glutamine, 200 units/mL penicillin and 200 μg/mL streptomycin (all from Sigma-Aldrich) and 10% FBS (Biowest), till 70% destruction of host cell monolayer. All the contents of the flask (the adherent cells collected using a cell scraper and the culture supernatants) were centrifuged at 1500×g for 20 min. The pellet was then passed through a 25G needle and washed three times in complete RPMI medium (all centrifugations were done at 1500×g for 20 min). The final pellet was resuspended in 3 mL of complete RPMI medium and passed through a PD-10 column filled with Sephadex G-25 M (GE Healthcare Life- Sciences, Freiburg, Germany). Freeze-killed NcT were prepared from suspensions of live tachyzoites (prepared as described above) resuspended in complete RPMI and kept at least one week frozen at − 80 °C, since others have previously shown that a 2 h freezing incubation at − 70 °C was enough to inactivate NcT^{33 (link)}.