Cell disruption

T. cruzi (X10/7 strain) mid-log-phase epimastigotes (∼1 × 10¹⁰) were harvested by centrifugation (1,912 × g, 15 min, 4°C) and washed with ice-cold PBS (1,912 × g, 5 min, 4°C); finally, the cell pellet was resuspended in 8 ml of ice-cold lysis buffer (1 mM EDTA, 1 mM dithiothreitol [DTT], 100 μM TLCK [Nα-p-tosyl-l-lysine chloromethyl ketone], and 1× Roche EDTA-free COmplete protease inhibitor cocktail in 50 mM potassium phosphate buffer [pH 7.4]). The cell suspension was submitted to three freeze-thaw cycles in a dry ice/ethanol bath to biologically inactivate the parasites and then submitted to cell disruption (Constant Systems, UK) at 30 kpsi. The resulting lysate was centrifuged (100,000 × g, 20 min, 4°C), the supernatant was collected, and the protein concentration was determined using a Bio-Rad protein assay.

L. donovani promastigotes WT and transgenic cell lines confirmed as overexpressing OSC and HP were grown for 72 h in roller bottles, starting at an initial concentration of 1 × 10⁵ cells mL⁻¹ (1.5 × 10⁷ cells in total). Mid-log phase promastigotes were washed with ice-cold PBS and harvested by centrifugation (1912 g, 15 min, 4°C). The cell pellets were resuspended in 8 mL of ice-cold lysis buffer (1 mM EDTA, 1 mM DTT, 100 μM TLCK, and 1× Roche EDTA-free cOmplete protease inhibitor cocktail in 50 mM potassium phosphate buffer, pH 7.4), submitted to 3 freeze−thaw cycles in a dry ice/ethanol bath to biologically inactivate the parasites and followed by cell disruption (Constant Systems, UK) at 30 kpsi. The resulting lysates were centrifuged (100,000 g, 20 min, 4°C), supernatants were collected, and the protein concentrations were determined using the Bio-Rad Protein Assay.