Preparation of T. cruzi Soluble Antigens

The T. cruzi soluble antigens (TcSA) were obtained from the Y strain using previously described methods (Martinez-Calvillo et al., 2007 (link); Fernandez-Villegas et al., 2011 (link); Mateus et al., 2013 (link)). Briefly, amastigotes and trypomastigotes (1:1 ratio) were collected from the VERO cell culture supernatants at 96-120 hours postinfection (Fernandez-Villegas et al., 2011 (link)). The parasites were then washed twice with cold 1× PBS (Eurobio) and resuspended at a density of 1 x 10⁶ parasites/μl in lysis buffer as previously reported (Martinez-Calvillo et al., 2007 (link)). The parasites were incubated on ice for 30 minutes, and supernatants containing TcSA were collected by centrifugation at 12,000 x g and 4°C for 15 minutes and stored at -80°C until use. The protein concentrations were determined using the Bradford assay, and the protein profiles were analyzed by SDS-PAGE followed by Coomassie blue staining (Gibco BRL; Grand Island, NY, USA). Noninfected VERO cell supernatants were subjected to all above-described procedures and used as a mock control in the flow cytometry assays.