Cysteine Protease Activity Quantification

Evaluation of the intracellular cysteine protease activity in total amebic lysates of treated trophozoites with E-64, ACh 1, 0.01, 0.0001, or 0.000001 µM as previously described (Bosch et al., 2012 (link); Dolabella et al., 2012 (link)). 100 µg of amebic extract and 2 mg of azo dye-impregnated collagen (Sigma-Aldrich, St Louis, Missouri, USA) resuspended in 500 µL of protease activation buffer (100 mM Tris pH 7.0 and 10 mM CaCl2), were incubated at 37°C for 18 h. After this reaction was stopped by adding 500 µL of 10% TCA. Then samples were centrifuged, collagen fibers were discarded, and supernatants collected for spectrophotometric determination at 540 nm.